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WO2024187091A1 - Compositions et procédés d'amplification isotherme d'acides nucléiques - Google Patents

Compositions et procédés d'amplification isotherme d'acides nucléiques Download PDF

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WO2024187091A1
WO2024187091A1 PCT/US2024/019074 US2024019074W WO2024187091A1 WO 2024187091 A1 WO2024187091 A1 WO 2024187091A1 US 2024019074 W US2024019074 W US 2024019074W WO 2024187091 A1 WO2024187091 A1 WO 2024187091A1
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Prior art keywords
amplicon
nucleic acid
sequence
polymerase
region
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Courtney DAILLEY
Anindita ROY
Mohammad ROKY
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Seek Labs Inc
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Seek Labs Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • PCR Polymerase chain reaction
  • CRISDA CRISPR-Cas9 triggered nicking endonuclease mediated strand displacement amplification
  • Cas9AR Cas9-nickase based Amplification Reaction
  • CRISDA uses a combination of reaction enzymes including Cas, a nicking restriction endonuclease, and a DNA polymerase, all of which need to be optimized in a single reaction mix.
  • Cas9AR uses a single Cas enzyme for initial priming and extension of target sequences.
  • Both CRISDA and Cas9AR are subjected to ssDNA amplification products blocking primer binding regions or primers inhibiting ssDNA cut sites which can inhibit amplification efficiency and, in some instances, prevent exponential amplification.
  • Transcription-based isothermal amplification methods have also been developed including Transcription-Mediated Amplification (TMA), Self-Sustained Sequence Amplification Reaction (3SR) and Nucleic Acid Sequence-based Amplification (NASBA). These methods can require steps such as heat WSGR Docket No. 58557-707.601 denaturation of dsDNA template and use of enzymes including Reverse Transcriptase (RT) and RNase H.
  • NAA nucleic acid amplification
  • PoN point-of-need
  • Existing methods can require temperature cycling during amplification reactions, or maintenance of reaction temperature within specific defined ranges which may not be conducive to operating at a given ambient temperature at a particular location.
  • isothermal NAA methods have been developed recently and these methods can suffer from lack of versatility to be applied in a wide range of PoN settings.
  • Previous isothermal amplification methods cannot operate effectively or efficiently at ambient temperatures in a broad range of PoN environments in which targeted nucleic acid amplification is desired.
  • Previous isothermal amplification methods may require reaction temperatures to be maintained at a constant temperature using an incubator. This creates difficulty in operating these methods in a resource-limited setting. Previous isothermal amplification methods may require complicated experimental design hindering their utility. Previous isothermal amplification methods may create double stranded DNA (dsDNA) amplification products which can be difficult to identify and quantify. dsDNA products are not amenable to binding to a diagnostic probe without additional method steps of preparation such as denaturation or enzymatic steps. Previous isothermal amplification methods may limit amplification rate of single stranded DNA (ssDNA).
  • ssDNA single stranded DNA
  • PoN NAA Technology To meet the WHO’s ASSURED (Affordable, Sensitive, Specific, User-Friendly, Rapid and Robust, Equipment-free, Deliverable) guidelines for a PoN NAA Technology (NAAT), there is an urgent need for improved methods of nucleic acid amplification. Desired improvements to PoN NAAT include i) simplicity of design, ii) effective and efficient reactions that can be carried out at any room temperature, iii) ability to detect specific amplification products for probe-based detection without the use of expensive or complicated instrumentation, iv) reactions with high specificity and sensitivity, and v) reactions that are easily transferrable to a variety of detection platforms.
  • CRISPR-assisted Isothermal Nucleotide Amplification Described herein are methods of CRISPR-assisted Isothermal Nucleotide Amplification involving introducing a targeted double strand break (DSB) and a targeted single strand break WSGR Docket No. 58557-707.601 (nick) near each other in a target nucleic acid sequence at the same time or using sequential steps.
  • DSB targeted double strand break
  • WSGR Docket No. 58557-707.601 nick
  • the method of claim 1, comprising a step of: iv) exposing one or a plurality of the first amplicons to: (a) a first primer, wherein the first primer designated (P1v1) comprises a domain complementary to the first amplicon; or wherein the first primer designated (P1v2) comprises a first domain complementary to the first amplicon, a second domain complementary to a first sgRNA target sequence, and a third domain complementary to a first PAM sequence; or wherein the first primer designated (P1v3) comprises a first domain complementary to the first amplicon, a second domain complementary to a first sgRNA target sequence, a third domain complementary to a first PAM sequence, and a fourth domain containing a 5’ extension; or wherein the first primer designated (P1v4) comprises a first domain complementary to a second sgRNA target sequence and a second domain complementary to a second PAM sequence, both of which are complementary to the first amplicon; and (b) a polymerase or functional fragment thereof in the
  • the method comprises steps of: v) exposing the second amplicon to the first editing enzyme and the first guide RNA for a time period sufficient to create a single-stranded break of the nucleic acid sequence at the second domain; or exposing the second amplicon to the first editing enzyme and a second guide RNA for a time period sufficient to create a single-stranded break of the second WSGR Docket No.
  • the first guide RNA comprises at least one domain complementary to the first region of the nucleic acid sequence, wherein, at step (iv), the first guide RNA binds to the third domain of the second amplicon; (vi) extending a 3’ end of the single-stranded break of the second amplicon and create a third nucleic acid sequence complementary to a single strand of the amplification target sequence by exposing a polymerase or functional fragment thereof in the presence of dNTPs for a time period sufficient for the polymerase or functional fragment thereof to extend, thereby generating a third amplicon by strand displacement action, wherein the first amplicon and the third amplicon are newly synthesized, single-stranded copies of both strands of the amplification target sequence; and (vii) repeating step (iv)-(vi) for a time period until a plurality of third amplicons are generated.
  • the first primer (P1v1), in the 5’ to 3’ orientation comprises the first domain.
  • the first primer (P1v2), in the 5’ to 3’ orientation comprises the third domain, the second domain, and the first domain.
  • the first primer (P1v3), in the 5’ to 3’ orientation comprises the fourth domain, third domain, the second domain, and the first domain.
  • the first primer (P1v4), in the 5’ to 3’ orientation comprises the first domain and the second domain.
  • the first primer further comprises an RNA polymerase promoter sequence positioned between the first and third domains.
  • the RNA polymerase promoter sequence comprises a T7 RNA polymerase promoter sequence, a T3 RNA polymerase promoter sequence, or an SP6 RNA polymerase promoter sequence.
  • the method comprises a step of: (viii) exposing the second amplicon to an RNA polymerase or functional fragment thereof and dNTPs comprising ribonucleic acid or a derivative thereof for a time period sufficient to generate a third amplicon that is a single-stranded RNA sequence complementary to the second amplicon.
  • the method comprises steps of: (ix) exposing one or a plurality of the third amplicons to: (a) a second primer, the second primer comprising a first domain complementary to the third amplicon, a second domain complementary to an sgRNA target sequence, and a third domain complementary to a PAM sequence, or the second primer comprising a domain complementary to the third amplicon, or the second primer comprising a first domain complementary to an sgRNA target sequence and a second domain complementary to a PAM sequence; and (b) a polymerase or functional fragment thereof in the presence of dNTPs; in each case, for a time period sufficient for the polymerase or functional fragment thereof to create complementary strand to the third amplicon thereby generating a fourth amplicon, wherein the fourth amplicon is a double-stranded copy of the amplification target sequence; (x) exposing the fourth amplicon to the first editing enzyme and the first guide RNA for a time period sufficient to create a single-stranded
  • the first editing enzyme is a Cas protein.
  • the Cas protein is chosen from: Cas9, Cas12, Cas12a or a functional fragment thereof.
  • the Cas9 protein is Cas9n D10A or Cas9n H840A.
  • the Cas12a protein is LbCas12a or SmCas12a.
  • the first region is positioned at or proximate to the 5’ end of the amplification target sequence and wherein the second region is positioned at or proximate to the 3’ end of the amplification target sequence.
  • the first region and the second region flank the 5’ end of the amplification target sequence and the 3’ end of the amplification target sequence, respectively. In some embodiments, the first region and the second region contiguously flank the 5’ end of the amplification target sequence and the 3’ end of the amplification target sequence, respectively. In some embodiments, the steps are performed at a temperature from about 20 degrees Celsius to about 42 degrees Celsius. In some embodiments, the steps are performed at a temperature from about 25 degrees Celsius to about 37 degrees Celsius. In some embodiments, the steps are performed at a temperature from about 25 degrees Celsius to about 45 degrees Celsius. In some embodiments, the steps are performed at approximate isothermal temperature. In some embodiments, the reaction is performed for a range of 5 minutes to 60 minutes.
  • the reaction is performed for about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes.
  • the reaction mixture comprises a labelled primer or a gene-specific probe such as a molecular beacon, which can hybridize to an amplification product and can be detected using a variety of methods such as lateral flow assay, fluorescence assay, electrochemical sensors, chemiluminescence assay, electrophoresis gels, or any combination thereof.
  • the reaction mixture comprises a DNA binding dye such as SYBR TM Green or methylene blue which can bind to WSGR Docket No.
  • ssDNA amplicon is detected by LFA.
  • one or more ssDNA amplicon is detected and quantified by LFA.
  • primer length is optimized to achieve efficient target amplicon production. In some embodiments, optimized primer length between about 15-70 nt. In some embodiments, optimized primer length between about 20-27 nt. In some embodiments, optimized primer length between about 27-36 nt.
  • the method comprises adding a single- strand binding protein to the reaction mix to increase target amplicon production efficiency.
  • the single-strand binding protein comprises gp32.
  • primers used for amplification comprise a FAM label.
  • the FAM-labeled primers is a nicking primer capable of binding on nicking sites to regenerating nick sites.
  • an amplicon comprising one or more FAM-labeled primers can be detected by direct FAM fluorescence channel.
  • Described herein are methods of CRISPR-assisted Isothermal Nucleotide Amplification involving introducing at least two targeted DSBs and at least two targeted nicks near each other in a target nucleic acid sequence at the same time or using sequential steps.
  • nucleic acid sequence comprising an amplification target sequence
  • the nucleic acid sequence comprising a first region, a second region, a third region and a fourth region that are positioned downstream from each other, with the first and second region being at or proximate to one end of the amplification target sequence, and the third and fourth region on the other end of the amplification target sequence
  • the method comprising: (i) exposing the nucleic acid sequence to a first editing enzyme or a functional fragment thereof and (a) a first guide RNA for a time period sufficient to create a double- stranded break of the nucleic acid sequence at or proximate to the first region, and (b) a second guide RNA for a time period sufficient to create a double-stranded break of the nucleic acid sequence at or proximate to the fourth region, effectively cutting out the second and third region comprising the amplification target sequence.
  • the method comprises steps: (ii) exposing the nucleic acid sequence to a second editing enzyme or a functional fragment thereof and a third guide RNA to create single-stranded break of the nucleic acid sequence at the nick site 1 on or proximate to the second region or the amplification target sequence; and exposing the nucleic acid sequence to a polymerase or functional fragment thereof in the presence of dNTPs for a time period sufficient for the polymerase or functional fragment thereof to extend the 3’ end of the single stranded break and create a second nucleic WSGR Docket No.
  • the method comprises a step: (v) exposing one or a plurality of the second amplicons to: (a) a first primer, wherein the first primer designated (P1v1) comprises a domain complementary to the first amplicon; or wherein the first primer designated (P1v2) comprises a first domain complementary to the first amplicon, a second domain complementary to an sgRNA target sequence, and a third domain complementary to a PAM sequence; or wherein the first primer designated (P1v3) comprises a first domain complementary to the first amplicon, a second domain complementary to an sgRNA target sequence, a third domain complementary to a PAM sequence, and a fourth domain containing a 5’ extension; or wherein the first primer designated (P1v4) comprises a first domain complementary to an sgRNA target sequence and a second domain complementary to a PAM sequence, both of which are complementary to the first amplicon; and (b) a polymerase or functional fragment thereof in the presence of dNTPs; in each case
  • the method comprises steps: (vi) exposing the double stranded amplicon to the second editing enzyme and the third guide RNA for a time period sufficient to create a single- stranded break of the nucleic acid sequence, wherein the third guide RNA comprises at least one domain complementary to the second region of the nucleic acid sequence; (vii) extending the 3’ end of the single-stranded break of the double stranded amplicon and creating a complementary nucleic acid sequence by exposing a polymerase or functional fragment thereof in the presence of dNTPs for a time period sufficient for the polymerase or functional fragment thereof to WSGR Docket No.
  • 58557-707.601 extend, thereby generating a third amplicon by strand displacement action, wherein the third amplicon is a newly synthesized, single-stranded copy of the first amplicon and the amplification target sequence; and (viii) repeating step (v)-(vii) for a time period until a plurality of third amplicons are generated; thereby resulting in an exponential accumulation of a single stranded copy of the target sequence.
  • the method comprises steps: (ix) exposing one or a plurality of the first amplicons to: (a) a second primer, the second primer comprising a domain complementary to the second amplicon, or wherein the second primer comprises a first domain complementary to the second amplicon, a second domain complementary to an sgRNA target sequence, a third domain complementary to a PAM sequence, or wherein the second primer comprises of a first domain complementary to the second amplicon, a second domain complementary to an sgRNA target sequence, a third domain complementary to a PAM sequence and a fourth domain containing a 5' extension, or wherein a second primer consists of a first domain complementary to the second amplicon, and a second domain complementary to nicking endonuclease restriction site; and (b) a polymerase or functional fragment thereof in the presence of dNTPs; in each case, for a time period sufficient for the polymerase or functional fragment thereof to create complementary strand to the first amplicon thereby generating
  • the first editing enzyme is a Cas enzyme, restriction endonuclease or nicking endonuclease.
  • the first Cas enzyme is selected from: Cas9, Cas12, Cas12a or a functional fragment thereof.
  • the first Cas protein is LbCas12a or SmCas12a.
  • the first Cas protein is Cas9n D10A or Cas9n H840A.
  • the second editing enzyme is a Cas enzyme, restriction endonuclease or nicking endonuclease. In some WSGR Docket No.
  • the second Cas enzyme is chosen from: Cas9, Cas12, Cas12a or a functional fragment thereof, a restriction endonuclease, nicking endonuclease or functional fragment thereof.
  • the second Cas protein is LbCas12a or SmCas12a.
  • the second Cas protein is Cas9n D10A or Cas9n H840A. In some embodiments, only a single editing enzyme is used.
  • the first region is positioned at or proximate to the 5’ end of the second region and wherein the fourth region is positioned at or proximate to the 3’ end of the third region.
  • the second region is positioned at or proximate to the 5’ end of the amplification target sequence and wherein the third region is positioned at or proximate to the 3’ end of the amplification target sequence.
  • the first region is positioned at or proximate to the 3’ end of the second region and wherein the fourth region is positioned at or proximate to the 5’ end of the third region.
  • the second region is positioned at or proximate to the 3’ end of the amplification target sequence and wherein the third region is positioned at or proximate to the 5’ end of the amplification target sequence.
  • the second region and the third region flank the 5’ end of the amplification target sequence and the 3’ end of the amplification target sequence, respectively. In some embodiments, the second region and the third region contiguously flank the 5’ end of the amplification target sequence and the 3’ end of the amplification target sequence, respectively. In some embodiments, the steps are performed at a temperature from about 20 degrees Celsius to about 42 degrees Celsius. In some embodiments, the steps are performed at a temperature from about 20 degrees Celsius to about 30 degrees Celsius. In some embodiments, the steps are performed at a temperature from about 20 degrees Celsius to about 25 degrees Celsius. In some embodiments, the steps are performed at approximate isothermal temperature.
  • the steps comprising a reaction are performed for a range of about 5 minutes to about 60 minutes. In some embodiments, the reaction is performed for about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes.
  • the reaction comprises a mixture comprising a labelled primer or a gene-specific probe such as a molecular beacon, which can hybridize to one or more amplification products of the reaction and can be detected using a variety of methods such as lateral flow assay, fluorescence assay, electrochemical sensors, chemiluminescence assay, electrophoresis gel, or any combination thereof.
  • the reaction comprises a mixture comprising a DNA binding dye such as SYBR TM Green or methylene blue which can bind to one of more amplification products of the reaction and can be detected using a variety of methods such as lateral flow assay, fluorescence assay, electrochemical sensors, chemiluminescence assay, electrophoresis WSGR Docket No. 58557-707.601 gel, or any combination thereof.
  • a DNA binding dye such as SYBR TM Green or methylene blue which can bind to one of more amplification products of the reaction and can be detected using a variety of methods such as lateral flow assay, fluorescence assay, electrochemical sensors, chemiluminescence assay, electrophoresis WSGR Docket No. 58557-707.601 gel, or any combination thereof.
  • one or more ssDNA amplicon is detected by LFA.
  • one or more ssDNA amplicon is detected and quantified by LFA.
  • primer length is optimized to achieve efficient target
  • optimized primer length between about 20-27 nt. In some embodiments, optimized primer length between about 27-36 nt. In some embodiments, optimized primer length between about 36-55 nt. In some embodiments, the method comprises adding a single- strand binding protein to the reaction mix to increase target amplicon production efficiency. In some embodiments, the single-strand binding protein comprises gp32. In some embodiments, primers used for amplification comprise a FAM label. In some embodiments, the FAM-labeled primers is a nicking primer capable of binding on nicking sites to regenerating nick sites. In some embodiments, an amplicon comprising one or more FAM-labeled primers can be detected by direct FAM fluorescence channel.
  • nucleic acid sequence comprising an amplification target sequence, the nucleic acid sequence comprising a first region, and a second region, that are positioned downstream from each other, with the method comprising: (i) exposing the nucleic acid sequence to a first editing enzyme or a functional fragment thereof and a first guide RNA to create single-stranded break of the nucleic acid sequence at the nick site 1 on or proximate to the first region or the amplification target sequence; and exposing the nucleic acid sequence to a polymerase or functional fragment thereof in the presence of dNTPs for a time period sufficient for the polymerase or functional fragment thereof to extend the 3’ end of the single stranded break and create a second nucleic acid sequence complementary to a single strand of the amplification target sequence, thereby generating a first amplicon via strand displacement action of the polymerase and regenerating the nick site 1 on the original target strand; (i)
  • the method comprises a step: (v) exposing one or a plurality of the second amplicons to: (a) a first primer, wherein the first primer designated (P1v1) comprises a domain complementary to the first amplicon; or wherein the first primer designated (P1v2) comprises a first domain complementary to the first amplicon, a second domain complementary to an sgRNA target sequence, and a third domain complementary to a PAM sequence; or wherein the first primer designated (P1v3) comprises a first domain complementary to the first amplicon, a second domain complementary to an sgRNA target sequence, a third domain complementary to a PAM sequence, and a fourth domain containing a 5’ extension; or wherein the first primer designated (P1v4) comprises a first domain complementary to an sgRNA target sequence and a second domain complementary to a PAM sequence, both of which
  • the method comprises steps: (vi) exposing the double stranded amplicon to the first editing enzyme and the first guide RNA for a time period sufficient to create a single-stranded break of the nucleic acid sequence, wherein the first guide RNA comprises at least one domain complementary to the second region of the nucleic acid sequence; (vii) extending the 3’ end of the single-stranded break of the double stranded amplicon and creating a complementary nucleic acid sequence by exposing a polymerase or functional fragment thereof in the presence of dNTPs for a time period sufficient for the polymerase or functional fragment thereof to extend, thereby generating a third amplicon by strand displacement action, wherein the third amplicon is a newly synthesized, single- stranded copy of the first amplicon and the amplification target sequence; and (viii) repeating step (v)-(vii) for a time period until a plurality of third amplicons are generated; thereby resulting in an exponential accumulation of
  • the method comprises steps: (ix) exposing one or a plurality of the first amplicons to: (a) a second primer, the second primer comprising a domain complementary to the second amplicon; and (b) a polymerase or functional fragment thereof in the presence of dNTPs; in each case, for a time period sufficient for the polymerase or functional fragment thereof to create complementary strand to the first amplicon thereby generating a double stranded amplicon, wherein the double stranded amplicon is a copy of the amplification target sequence; (x) exposing the double stranded amplicon to the first editing enzyme and the second guide RNA for a time period sufficient to create a single-stranded break of the nucleic acid sequence, WSGR Docket No.
  • the second guide RNA comprises at least one domain complementary to the second region of the nucleic acid sequence; (xi) extending the 3’ end of the single-stranded break of the double stranded amplicon and creating a complementary nucleic acid sequence by exposing a polymerase or functional fragment thereof in the presence of dNTPs for a time period sufficient for the polymerase or functional fragment thereof to extend, thereby generating a fourth amplicon by strand displacement action, wherein the fourth amplicon is the exact single- stranded complement to the third amplicon, wherein the third and fourth amplicon can bind each other to give a double stranded amplicon which is a copy of the target nucleic acid sequence; and (xii) repeating step (ix)-(xi) for a time period until a plurality of fourth amplicons are generated; thereby resulting in an exponential accumulation of a double stranded copy of the target sequence.
  • the first editing enzyme is a Cas enzyme, restriction endonuclease or nicking endonuclease.
  • the first Cas enzyme is selected from: Cas9, Cas12, Cas12a or a functional fragment thereof.
  • the first Cas protein is Cas9n D10A or Cas9n H840A.
  • the steps are performed at a temperature from about 20 degrees Celsius to about 42 degrees Celsius. In some embodiments, the steps are performed at a temperature from about 20 degrees Celsius to about 30 degrees Celsius. In some embodiments, the steps are performed at a temperature from about 20 degrees Celsius to about 25 degrees Celsius. In some embodiments, the steps are performed at approximate isothermal temperature.
  • the steps comprising a reaction are performed for a range of about 5 minutes to about 60 minutes. In some embodiments, the reaction is performed for about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes.
  • the reaction comprises a mixture comprising a labelled primer or a gene-specific probe such as a molecular beacon, which can hybridize to one or more amplification products of the reaction and can be detected using a variety of methods such as lateral flow assay, fluorescence assay, electrochemical sensors, chemiluminescence assay, electrophoresis gel, or any combination thereof.
  • the reaction comprises a mixture comprising a DNA binding dye such as SYBR TM Green or methylene blue which can bind to one of more amplification products of the reaction and can be detected using a variety of methods such as lateral flow assay, fluorescence assay, electrochemical sensors, chemiluminescence assay, electrophoresis gel, or any combination thereof.
  • a DNA binding dye such as SYBR TM Green or methylene blue which can bind to one of more amplification products of the reaction and can be detected using a variety of methods such as lateral flow assay, fluorescence assay, electrochemical sensors, chemiluminescence assay, electrophoresis gel, or any combination thereof.
  • one or more ssDNA amplicon is detected by LFA.
  • one or more ssDNA amplicon is detected and quantified by LFA.
  • primer length is optimized to achieve efficient target amplicon production. In some embodiments, optimized primer length between about 15- 70 nt. In some embodiments, optimized primer length between
  • optimized primer length between about 27-36 nt. In some embodiments, optimized primer length between about 36-55 nt. In some embodiments, the method comprises adding a single-strand binding protein to the reaction mix to increase target amplicon production efficiency. In some embodiments, the single-strand binding protein comprises gp32. In some embodiments, primers used for amplification comprise a FAM label. In some embodiments, the FAM-labeled primers is a nicking primer capable of binding on nicking sites to regenerating nick sites. In some embodiments, an amplicon comprising one or more FAM-labeled primers can be detected by direct FAM fluorescence channel.
  • FIG.1 depicts diagrams of genomic DNA (gDNA) template and amplification target features, single guide RNA (sgRNA)-targeted CRISPR associated protein (Cas) endonuclease digestion step and resulting single strand break (nick) and double strand break (DSB) in Strategy 1, version 1 of CRISPR-assisted isothermal nucleotide amplification (CINA).
  • sgRNA single guide RNA
  • Cas CRISPR associated protein
  • nick single strand break
  • DSB double strand break
  • FIG.2 depicts diagrams of gDNA template and amplification target features, sgRNA- targeted Cas endonuclease digestion step, and resulting nick in one section of target sequence and resulting two staggered nicks on complementary strands in another section of the target sequence in Strategy 1, version 2 of CINA. The two staggered nicks equate to one DSB with sticky ends.
  • FIG.3 shows two images of agarose gel products (1.5% agarose gel, run at 160V for 50 min) analyzed by electrophoresis and nucleic acid labeling.
  • FIG.4A shows in image of an agarose gel of nicked pUC19 plasmid and amplification of plasmid sequence of various DNA polymerases.
  • FIG.4B shows a graph a band density from Lane K and Lane B in FIG.4A indicating the extent of greater amplification from Bst 2.0 DNA polymerase compared to Klenow Fragment.
  • WSGR Docket No. 58557-707.601 FIG.5 depicts PART A of Strategy 1 of CINA. Illustrated are steps of nicking, nucleotide extension from nicked Nick-Site 1 on a forward strand, and linear amplification of single strand DNA (ssDNA) target sequence termed Amplicon 1.
  • ssDNA single strand DNA
  • FIG.6 depicts PART B1 of Strategy 1 of CINA. Illustrated are steps of Primer 1 (P1) binding to Amplicon 1 from PART A of Strategy 1 of CINA, nucleotide extension of 3’ ends of complementary strands to form a Regenerated Nick-Site 1 now on a reverse strand, nicking, nucleotide extension from nicked Nick-Site 1 on a reverse strand, and linear amplification of ssDNA target sequence termed Amplicon 2.
  • FIG.7 depicts PART B 2 of Strategy 1 of CINA. Modified Primer 1 (MP1) comprising a promoter sequence is illustrated binding to Amplicon 1 from PART A of Strategy 1 of CINA.
  • Nucleotide extension of 3’ ends of complementary strands is depicted to form a Regenerated Nick-Site 1 on a reverse strand adjacent to the promoter sequence.
  • RNA polymerase is depicted binding to the promoter sequence and synthesizing a single strand RNA (ssRNA) transcript of Amplicon 1 (termed Ramplicon 1) through linear amplification.
  • FIG.8 depicts PART C of Strategy 1 of CINA.
  • Primer 2 (P2) comprising part of Nick- Site 1 and a complimentary sequence for protospacer adjacent motif 1 (PAM1) is illustrated binding to Amplicon 2.
  • P2 comprising part of Nick- Site 1
  • PAM1 protospacer adjacent motif 1
  • nucleotide extension of 3’ ends of complementary strands to form a regenerated Nick-Site 1 on the forward strand.
  • FIG.9A shows primer design that can be used in CINA Strategy 1 and CINA Strategy 2.
  • FIG.9B depicts a diagram of a region of puc19 plasmid DNA sequence designed for CINA amplification using Strategy 1 or Strategy 2.
  • FIG.10 depicts DNA sequences of puc19 and location of CINA elements for Strategy 1 amplification including Nick Site 1, DSB1 for Amplicon 1, and Primer 1 for Amplicon 1.
  • FIG.11 depicts DNA sequences of puc19and location of CINA elements for Strategy 1 amplification including dsDNA 1 generated from Primer 1 extension via Amplicon 1, Amplicon 2, Nick Site for Cas9n, and NGG PAM location.
  • FIG.12 depicts DNA sequences of puc19 and location of CINA elements for Strategy 1 amplification including Amplicon 2 generated from nicking bottom strand dsDNA1, Primer 2 for Amplicon 2, and dsDNA2 generated from Primer 2 extension via Amplicon 2.
  • FIG.13 depicts DNA sequences of puc19 and location of CINA elements for Strategy 1 amplification including Amplicon 3 generated from Cas9n nicking the 5’ end non-sense strand of dsDNA2.
  • FIG.14 shows agarose gel (left) and LFA (right) results of CINA reaction products under various test conditions.
  • FIG.15A shows agarose gel (left), 10% Native PAGE (center), and LFA (right) results of CINA amplicons generated using both nicking & non-nicking primers for strategy 1 using the starting template from pUC19.
  • FIG.15B shows an image of the agarose gel in FIG.15A (left) and band quantification of reaction products from CINA methods in the chart on the right.
  • FIG.16 depicts diagrams of gDNA template and amplification target features, sgRNA- targeted Cas endonuclease digestion step, and resulting two DSBs in Strategy 2 of CRISPR- assisted isothermal nucleotide amplification (CINA) to isolate target sequence.
  • FIG.17 depicts sgRNA-targeted Cas endonuclease nicking of isolated target sequence in Strategy 2 of CINA.
  • FIG.18 depicts PART A of Strategy 2 of CINA.
  • FIG.19 depicts PART B of Strategy 2 of CINA. Illustrated are configurations of ssDNA 1 and ssDNA 2 which have partial complementarity and may form a staggered duplex.
  • FIG.20 depicts a summary of steps for ssDNA target amplification using Strategy 2 of CINA combining PARTA and PART B.
  • FIG.21 depicts optional PART C of Strategy 2 of CINA.
  • Primer 2 (P2) is shown binding ssDNA 1 from Part A.
  • DNA polymerase is depicted extending the 3’ end of P2, regenerating Cas Nick-Site 2 and amplifying until Nick-Site 1.
  • sgRNA-guided Cas is illustrated nicking the regenerated Cas Nick-Site 2.
  • DNA polymerase is depicted entering the nicked site and extending the 3’ end, thereby regenerated the nick site for subsequent nicking and extension, WSGR Docket No. 58557-707.601 while displacing the bottom strand labelled ssDNA 4 for linear amplification.
  • FIG.22 depicts summary of steps for Strategy 2 of CINA combining Parts A + B + C to produce dsDNA amplified target sequence.
  • FIG.23 shows a diagram of a portion of pUC19 plasmid sequence and labeled sites for use in CINA Strategy 2.
  • FIG.24A shows images of CINA Strategy 2 reaction products run on 10% Native PAGE.
  • FIG.24B shows images of CINA Strategy 2 reaction products run on 1.5% agarose gel electrophoresis.
  • FIG.25 shows diagrams of a lateral flow assay (LFA) probe design to detect ssDNA 1 amplicons produced from CINA Strategy 2.
  • FIG.26A shows an image of an agarose gel showing CINA Strategy 2 Part A+ B + C amplified expected amplicons visualized on the gel.
  • FIG.26B shows an image of LFA analysis to detect products from reactions shown in FIG.26A.
  • FIG.26C shows an image of the gel from FIG.26A for band intensity quantification analysis.
  • FIG.26D shows a graph of adjusted band density of dsDNA 3 in L6 compared to ssDNA 3 from L5.
  • FIG.26E shows a graph of adjusted band density of four amplicon products from the CINA methods used with reactions from FIG.26A.
  • FIG.27 shows a diagram of a 790 bp region of pUC19 plasmid DNA sequence designed for CINA amplification.
  • FIG.28 shows CINA reaction products amplified using BST 3.0 run on an agarose gel.
  • FIG.29 shows CINA reaction products amplified using BST 2.0 run on an agarose gel (left) and analyzed by LFA (right) using ssDNA1 probe for detection of specific amplicon.
  • FIG.30 shows CINA reaction products amplified using BST 3.0 run on an agarose gel (left) and analyzed by LFA (right).
  • FIG.31 shows CINA reaction products amplified using three different sets of nick regenerating primer.
  • FIG.32 shows two gels comparing specific CINA reaction input component ratios and the effects on efficiency of amplicon amplification.
  • WSGR Docket No. 58557-707.601 [00051]
  • FIG.33A shows a diagram of a FAM-labelled primer design for use in CINA methods and detection of CINA amplified products.
  • FIG.33B shows FAM exposure of an agarose gel following electrophoresis to demonstrate direct visualization of two in-reaction labeled CINA amplification products.
  • FIG.33C shows FAM exposure and Gel Red post staining of an agarose gel following electrophoresis to demonstrate direct visualization and verification of CINA reaction products.
  • FIG.33D shows a graph of calculated amplified band intensity for the CINA reaction shown in FIG.33C.
  • FIG.34 shows an agarose gel image of CINA reaction results testing various primer lengths in amplification efficiency.
  • FIG.35 shows two agarose gel images of CINA reaction results testing various reaction temperatures for the effects on amplification efficiency.
  • FIG.36 shows an image of sgRNA:Cas9n:DNA ratios tested and products run on agarose gel.
  • FIG.37 shows gel and LFA analysis results on CINA reaction product. Agarose gel (left) and 10% Native PAGE (center) show relative intensity of various CINA reaction products according to primer design. LFA results (right) show that labeled probe specifically detects ssDNA 1.
  • FIG.38 depicts DNA sequences of puc19 and location of CINA elements for Strategy 2 amplification including locations for staggered DSB1 and staggered DSB2 created by Cas12a targeting, Can9n Nick 1 site, TTTN PAM location for Cas12a DSB, NGG PAM location for Cas9, crRNA target sequence for Cas12a DSB1 and Cas12a DSB2, sgRNA target sequence for Cas9n D10A Nick 1, and excised dsDNA following creation of DSB1 and DSB2.
  • FIG.39 depicts DNA sequences of puc19 and location of CINA elements for Strategy 2 amplification including label map for excised dsDNA following creation of DSB1 and DSB2 from Cas12a targeting.
  • FIG.40 depicts DNA sequences of puc19 and location of CINA elements for Strategy 2 amplification including ssDNA1 product produced following Nick1 creation via Cas9n D10A targeting and ssDNA2 produced following Nick2 creation.
  • FIG.41 depicts DNA sequences of puc19 and location of CINA elements for Strategy 2 amplification including Primer 1 sequence and location of annealing to Amplicon 1 as well as dsDNA2 product generated from Primer 1 extension via Amplicon 1.
  • FIG.42 depicts DNA sequences of puc19 and location of CINA elements for Strategy 2 amplification including ssDNA2 product (172 bases(b)) in length generated from Cas9n Nick 2 WSGR Docket No. 58557-707.601 on the bottom strand. Also shown is Primer 2 sequence and the staggered duplex formed from ssDNA1 and ssDNA2 annealing. [00064] FIG.43 depicts DNA sequences of puc19 and location of CINA elements for Strategy 2 amplification including ssDNA1 and ssDNA2 staggered duplex formation, Primer 1 V1 and Primer 1 V2 features, and location of Primer 1 annealed to ssDNA23’ end.
  • FIG.44A – FIG.44B depicts DNA sequences of puc19 and location of CINA elements for Strategy 2 amplification including dsDNA product produced after primer 1 V2 extension (FIG.44A) and ssDNA produced following Nick 3 generated and Primer 1 V2 extension (FIG. 44B).
  • FIG.45 depicts DNA sequences of puc19 and location of CINA elements for Strategy 2 amplification including ssDNA1 and ssDNA2 staggered duplex formation, Primer 1 annealed to ssDNA23’ end, and Primer 2 annealed to ssDNA13’ end.
  • FIG.46 depicts a diagram of a region of puc19 plasmid DNA sequence designed for CINA amplification using Strategy 2 including location of excised dsDNA, ssDNA 2 produced following creation of Nick 2, ssDNA1 following creating of Nick 1, and the location of Primer 1 for Strategy 2 that overlaps with the location of Primer 1 for Strategy 1.
  • the novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments.
  • DETAILED DESCRIPTION Overview Described herein are methods and compositions relating to amplification of nucleic acid sequences.
  • compositions for use in isothermal or near isothermal methods of CRISPR-assisted nucleic acid amplification.
  • the compositions comprise one or a plurality of Cas proteins or functional fragments thereof.
  • the compositions comprise one or a plurality of guide RNA molecules.
  • the compositions comprise one or a plurality of oligonucleotide primers.
  • the compositions comprise one or a plurality of DNA polymerases.
  • the compositions comprise one or a plurality of ssDNA or ssRNA binding proteins. In some embodiments, the compositions comprise one or a plurality of target nucleic acid sequences.
  • WSGR Docket No. 58557-707.601 The present disclosure relates to a composition comprising a nucleic acid sequence comprising a target amplification sequence, a first and second editing enzyme, a plurality of dNTPs, and one or a plurality of primers.
  • the DNA polymerase is a strand-displacing DNA polymerase or a functional fragment thereof.
  • the strand-displacing DNA polymerase is selected from the group consisting of: Polymerase I Klenow fragment, Bst polymerase, Phi-29 polymerase, and Bacillus subtilis Pol I (Bsu) polymerase.
  • the DNA polymerase has reduced or absent exonuclease activity.
  • the DNA polymerase comprises Klenow Fragment.
  • the DNA polymerase comprises Bst 2.0.
  • the DNA polymerase comprises Bst 3.0.
  • the DNA polymerase comprises Bst 2.0 and Bst 3.0.
  • the DNA polymerase comprises Klenow Fragment, Bst 2.0, Bst 3.0, or any combination thereof.
  • the strand- displacing DNA polymerase comprises Polymerase I Klenow fragment, Bst polymerase, Phi-29 polymerase, and Bacillus subtilis Pol I (Bsu) polymerase.
  • the compositions comprise a recombinase and single-stranded DNA-binding or RNA- binding protein.
  • the DNA polymerase has reduced or absent exonuclease activity.
  • Cas Proteins [00074] Another aspect of the disclosure relates to a CRISPR system comprising a modified CRISPR enzyme (or "Cas protein") or a nucleotide sequence encoding one or more Cas proteins.
  • Cas stands for “CRISPR-associated protein”. Any protein capable of enzymatic activity in cooperation with a guide sequence is a Cas protein.
  • one, or two, or three, or four, or five, or more Cas proteins are targeted to a particular nucleic acid sequence or sequences in proximity to a target amplification sequence.
  • the nucleic acid sequence or sequences to which a Cas protein is targeted are a DNA sequence or DNA sequences.
  • the DNA sequence or DNA sequences correspond to a genomic DNA sequence at a given location within a genome or genomic DNA sequences at given locations within a genome.
  • a Cas protein is an enzyme or a functional fragments of an enzyme capable of cleaving specific strands of nucleic acids (including DNA) that are complementary to a CRISPR sequence.
  • a Cas protein, or a functional fragment thereof may function as an endonuclease.
  • a Cas protein, or a functional fragment thereof may cleave both strands of a DNA at a particular location.
  • a 58557-707.601 protein uses a HNH domain to cleave a DNA strand complementary to a crRNA sequence.
  • a Cas protein uses a RuvC domain to cleave a DNA strand that is non- complementary to a crRNA sequence.
  • a Cas protein uses a HNH domain to cleave a DNA strand complementary to a crRNA sequence and a RuvC domain to cleave a DNA strand that is non-complementary to a crRNA sequence.
  • a Cas protein, or a functional fragment thereof may cleave both strands of a DNA at a particular location to create a blunt- ended DSB.
  • Cas9n An example of a Cas protein that cleaves in this format is Cas9n.
  • a Cas protein, or a functional fragment thereof may cleave both strands of a DNA at a particular location to create a staggered-ended DSB.
  • An example of a Cas protein that cleaves in this format leaving a staggered-ended DBS is Cas12a.
  • Cas12a can cleave a target DNA strand 18-23 nucleotides distal of a protospacer adjacent motif (PAM) recognition site. In some embodiments, this staggered cleavage can leave been 5’ overhands between 5-8 nucleotides.
  • PAM protospacer adjacent motif
  • a Cas protein, or a functional fragment thereof may cleave a targeted nucleic acid site on a single strand.
  • this creation of a single-stranded DNA break is a called nicking and the ssDNA break itself is called a nick.
  • an enzyme capable of catalyzing a reaction to create a nick is called a nickase.
  • the Cas protein capable of creating a targeted ssDNA nick is Cas9n D10A.
  • the Cas protein capable of creating a targeted ssDNA nick is Cas9n H840A.
  • CRISPR (acronym for clustered regularly interspaced short palindromic repeats) sequences are a family of DNA sequences found in the genomes of various organisms.
  • a homolog of a Cas protein is selected to create a targeted nick and designed for use with an sgRNA according to the corresponding PAM sequence for the selected Cas protein to correctly target nicking.
  • a homolog of a Cas protein is selected to create a DBS (with blunt ends or staggered ends) and designed for use with an sgRNA according to the corresponding PAM sequence for the selected Cas protein to correctly target DBS creation.
  • a Cas9 enzyme or a Cas12 enzyme with respective PAM (Protospacer Adjacent Motif) site is selected for use in a method described herein.
  • PAM Protospacer Adjacent Motif
  • Some exemplary Cas proteins and PAM sequences are listed below: 1.
  • Spy Cas9 or spCas9 (the most commonly used WT Cas9 enzyme from Streptococcus pyogenes): The location of the double strand break (DSB) is within the target sequence 3 bases from the 3’ NGG PAM.
  • the PAM sequence, NGG must follow the targeted region on the opposite strand of the DNA with respect to the region complementary sgRNA sequence. targets DNA using a guide RNA complementary to a site with a 3’ NGG protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • spCas9 Nickase D10A or Cas9n (variant of spCas9 that makes nicks instead of DSB, sold by New England Biolabs or NEB): a variant of spCas9 nuclease differing by a point mutation (D10A) in the RuvC nuclease domain, which enables it to nick, but not cleave (DSB), DNA.
  • Spy Cas9n targets DNA using a guide RNA complementary to a site with a 3’ NGG PAM, similar to original Spy Cas9.
  • Double-stranded DNA breaks can be generated with reduced off-target cleavage by targeting two sites in close proximity (generally 0-20 bp apart) and with PAMs facing outward to leave 5’ overhangs (in CINA we call it double nicking strategy).
  • spCas9nickase H840A protein variant of spCas9 that makes nicks instead of DSB, sold by Applied Biological materials: a variant of spCas9 nuclease with a H840A mutation in its HNH-like nuclease domain, which enables it to generate a single-stranded nick instead of a DSB.
  • PAM site is 3’ NGG similar to spCas9.
  • nicking by the H840 variant occurs on the same DNA strand as the PAM and the target sequence.
  • Double- stranded DNA breaks can be generated with reduced off-target cleavage by targeting two sites in close proximity (generally 0-20 bp apart) and with PAMs facing inward to leave 5’ overhangs (in CINA we call it double nicking strategy).
  • Spy dCas9 active variant of spCas9, available on NEB: dCas9 contains two point mutations, D10A and H840A, which inactivate its RuvC and HNH nuclease domains, respectively, making it an inactive mutant of spCas9 nuclease that retains programmable DNA binding activity.
  • PAM site is 3’ NGG similar to spCas9.
  • Spy Cas9 HF1 high-fidelity variant of Spy Cas9 made by NEB, makes DSB not nicks
  • DSB Streptococcus pyogenes with reduced non- specific DNA cleavage
  • SpRY Cas9 engineered no-PAM requiring variant of spCas9 made by NEB: an engineered, RNA-guided, DNA endonuclease that catalyzes site-specific cleavage (DSB). Targeting requires a ⁇ 100 nucleotide single guide RNA (sgRNA) with complementarity to the 20-nucleotide region immediately upstream of a protospacer adjacent motif (PAM) on the dsDNA substrate.
  • sgRNA nucleotide single guide RNA
  • PAM protospacer adjacent motif
  • DNA cleavage by EnGen SpRY Cas9 produces a double-stranded break occurring 3 nucleotides upstream of the PAM.
  • Sau Cas9 WT Cas9 from Staphylococcus aureus, available from NEB: RNA-guided DNA endonuclease that catalyzes site-specific cleavage (DSB) of double-stranded DNA.
  • DSP site-specific cleavage
  • DNA cleavage by EnGen Sau Cas9 occurs ⁇ 3 bases 5′ of the PAM. 4.
  • Seq1 Cas9 WT Cas9 from Streptococcus equinus, available from NEB: RNA-guided DNA endonuclease that catalyzes site-specific cleavage (DSB) of double-stranded DNA.
  • DLB site-specific cleavage
  • Targeting requires a 116-nucleotide single guide RNA (sgRNA).
  • the sgRNA encodes a 20-nucleotide sequence with complementarity to the DNA target strand immediately upstream of a 5 ⁇ – NAGA –3 ⁇ PAM on the non-target strand.
  • DNA cleavage by EnGen Seq1 Cas9 produces a double-stranded break occurring 3 nucleotides upstream of the PAM.
  • Lba Cas12a or lbCas12a or lbCpf1 WT Cas12a enzyme from Lachnospiraceae bacterium ND2006, available from NEB
  • WT Cas12a enzyme from Lachnospiraceae bacterium ND2006, available from NEB A site-specific DNA endonuclease guided by a single 41-44 nucleotide guide RNA (gRNA).
  • gRNA nucleotide guide RNA
  • Targeting requires a gRNA complementary to the target site as well as a 5’ TTTV PAM (V is A, C or G) on the DNA strand opposite the target sequence.
  • ftCas12a or ftCpf1 WT Cas12a enzyme from Francisella tularensis, available from Intact Genomics: A site-specific DNA endonuclease guided by a single 41-44 nucleotide guide RNA (gRNA). Targeting requires a gRNA complementary to the target site as well as a 5’ TTTV PAM (V is A, C or G) on the DNA strand opposite the target sequence.
  • smCas12a or smCpf1 WT Cas12a enzyme from Smithella sp., not available commercially: A site-specific DNA endonuclease guided by either a processed single 55-61 nucleotide guide RNA (gRNA), or an unprocessed single 37-44 nucleotide guide RNA (gRNA).
  • Targeting requires a gRNA complementary to the 20-24 nucleotide target site as well as a TTTV PAM (where V is A, C or G) on the DNA strand opposite the target sequence.
  • Cleavage by SmCas12a occurs 18 nucleotides of the PAM and leaves 5′ overhanging/sticky ended DSB.
  • smCas12a doesn’t have any trans cleavage activity.
  • the Cas protein, or functional fragment thereof may be a wild-type form of endonuclease.
  • the wild-type form of Cas protein comprising an endonuclease may be Cas9, Cas12, Cas12a (or Cpf1 or Mad7), Cas12b (or C2c1 or Cpf2), Cas12c (C2c3), Cas12d (or CasY), Cas12e (or CasX), Cas13, Cas13a (or C2c2), Cas13b (or C2c6), Cas13c (or C2c7), Cas13d (or Casrx), Cas14, Cas14a, Cas14b, Cas14c, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al , Cas8
  • the Cas protein, or functional fragment thereof may be a mutated Cas endonuclease.
  • the mutated Cas endonuclease is derived from wildtype endonucleases such as Cas9, Cas12, Cas12a (or Cpf1 or Mad7), Cas12b (or C2c1 or Cpf2), Cas12c (C2c3), Cas12d (or CasY), Cas12e (or CasX), Cas13, Cas13a (or C2c2), Cas13b (or C2c6), Cas13c (or C2c7), Cas13d (or Casrx), Cas14, Cas14a, Cas14b, Cas14c, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8
  • Cas9 protein is an enzyme harboring site-specific nuclease WSGR Docket No. 58557-707.601 activity.
  • Cas9 enzymes, and functional fragments thereof, contain two endonuclease domains; HNH and RuvC, which catalyze the cleavage of phosphodiester bonds between nucleic acids on the complementary and non-complementary strands of the target duplex.
  • the CRISPR enzyme is Cas9 and may be Cas9 from S. pyogenes or S. pneumoniae.
  • the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence.
  • the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 8, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, or more base pairs from the first or last nucleotide of a target sequence.
  • a vector encodes a CRISPR enzyme or Cas protein that is mutated to with respect to a corresponding wild-type enzyme, such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence.
  • an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand).
  • a Cas9 nickase may be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ.
  • a Cas protein is an engineered or mutated variant in order to increase reaction kinetics of DNA cleavage whose versions are herein incorporated by reference: i) (Gopalappa R, et al.
  • Paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption.
  • FrCas9 is a CRISPR/Cas9 system with high editing efficiency and fidelity.
  • engineered Cas variants increase specificity of targeted DNA cleavage, increase fidelity of target DNA cleavage, reduce off-target effects of DNA cleavage, or any combination thereof.
  • use of FrCas9 in compositions described herein increases a rate of spacer acquisition, increases DNA cleavage efficiency of targets with NAP PAMS, or a combination thereof.
  • use of FrCas9 in compositions described herein induces targeted mutations at high frequencies with limited off target effects.
  • Hyper-Cas9 comprising a substitution of at least one of the following amino acids: I473 and K500 of wild-type Cas9 in compositions described herein increases a rate of spacer WSGR Docket No. 58557-707.601 acquisition, increases DNA cleavage efficiency of targets with NAP PAMS, or a combination thereof.
  • I473F substitution in a Cas protein variant increases specificity for NAG flanked targets.
  • LbCas12a variants derived from Lachnospiraceae bacterium used in compositions described herein show a broad PAM preference by recognizing certain non-canonical PAMs with high efficiency.
  • an LbABE8e variant carrying G532R and/or K595R mutations alter the original PAM specificity.
  • the LbABE8e variant carrying G532R and/or K595R mutations exhibits superior base editing activity compared with wild-type LbABE8e at sites with non-canonical PAMs.
  • a LbCas12a variant carries the G532R and/or K595R mutations.
  • a Cas12a derived from Lachnospiraceae bacterium is used as the first Cas enzyme or the second Cas enzyme for targeted cutting of nucleotide sequence creating staggered cleavage.
  • a Cas12a derived from a Smithella species is used as the first Cas enzyme or the second Cas enzyme for targeted cutting of nucleotide sequence creating staggered cleavage.
  • two or more catalytic domains of Cas9 may be mutated to produce a mutated Cas9 substantially lacking all DNA cleavage activity.
  • a D10A mutation is combined with one or more of H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA cleavage activity.
  • a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or lower with respect to its non-mutated form.
  • Other mutations may be useful; where the Cas9 or other CRISPR enzyme is from a species other than S. pyogenes, mutations in corresponding amino acids may be made to achieve similar effects.
  • a guide RNA (gRNA) sequence comprising complementarity to at least a portion of target nucleic acid sequence.
  • the complementarity to at least a portion of target nucleic acid sequence may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 55, 60, 65, 70, or 75 nucleotides bases. Selection of a particular target sequence complement in the design of an sgRNA molecule allows for precise targeting of a Cas protein when combined with the sgRNA to a particular genomic site.
  • the sgRNA sequences comprise a portion of a linear nucleotide sequence unique within a selected genome.
  • the one or more sgRNA sequences comprise portions of a linear WSGR Docket No. 58557-707.601 nucleotide sequence complementary to one or more specific, defined positions within the genome.
  • the sgRNA sequences are non-coding sequences.
  • one or more dsDNA targets are amplified. In some embodiments, one or more ssDNA and dsDNA targets are amplified. In some embodiments, the one or more ssDNA and dsDNA amplified targets are referred to as amplicons. In some embodiments, amplified DNA as a reaction outcome of a CINA method produces copies of a target DNA sequence that resides within a template DNA region.
  • the amplified DNA product is produced during the reaction at a rate of approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0, 2.0, 3.0, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20, 22.5, 25, 27.5, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 98.5, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, 99.95, or 99.99 percent of a rate of linear amplification.
  • the amplified DNA product is produced during the reaction at a rate of near exponential amplification. In some embodiments, the amplified DNA product is produced during the reaction at a rate of exponential amplification.
  • CINA Strategy 1 [00082]
  • the methods introduce a DSB and a nick in proximity near each other in a linear portion of nucleic acid. In some embodiments, a DSB and a nick are used as part of CINA Strategy 1. The DSB and the nick may be introduced at the same time or sequentially. The DSB and the nick made sequentially may be made using sequential steps. In some embodiments, a Cas protein or functional fragment thereof creates a staggered DSB.
  • the staggered DSB is created by targeting a specific region of nucleic acid with Cas12a.
  • targeted Cas protein introduction of two staggered nicks equates to one DSB with one or two sticky ends.
  • a Cas protein or WSGR Docket No. 58557-707.601 functional fragment thereof creates a blunt DSB.
  • the blunt DSB is created by targeting a specific region of nucleic acid with Cas9n.
  • Appropriate sgRNAs are used to target one or more Cas proteins to a target region of interest near the sites of nucleic acid amplification.
  • a nick is created at a location in close linear proximity to a DSB.
  • a single Cas nickase can be targeted to multiple locations near a nucleic acid target of interest to create both a DSB1 site and a Nick Site 1.
  • two Cas proteins can be targeted to multiple locations near a nucleic acid target of interest to create both a DSB1 site and a Nick Site 1.
  • two or more Cas proteins can be targeted to multiple locations near a nucleic acid target of interest to create both a DSB1 site and a Nick Site 1.
  • target genomic DNA contains at least one PAM sequence for a specific Cas protein. In some embodiments, target genomic DNA (gDNA) contains at least two PAM sequences for one or more specific Cas proteins.
  • target genomic DNA contains at least three PAM sequences for one or more specific Cas proteins. In some embodiments, target genomic DNA (gDNA) contains at least four PAM sequences for one or more specific Cas proteins. In some embodiments, target genomic DNA (gDNA) contains at least five PAM sequences for one or more specific Cas proteins.
  • ssDNA 1 As the DNA polymerase extends, it displaces a ssDNA target sequence on the forward strand. By recreating Nick Site 1 in the presence of these enzymes and reaction conditions, ssDNA1 is amplified by linear amplification during the reaction. ssDNA 1 is referred to as Amplicon 1. WSGR Docket No. 58557-707.601 [00084]
  • Primer 1 (P1) when ssDNA 1 is produced, has a section that is complementary to the 3’ end of ssDNA 1 and can hybridize to Amplicon 1. Upstream of the complementary hybridization site, P1 further comprises a complement sequence for PAM1 and a Cas Nick Site 1 sequence.
  • Binding of P1 to Amplicon 1 allows DNA polymerase to extend each strand in the 3’ direction to end points defined by the 5’ end of P1 and the 5’ end of Nick Site 1.
  • Nick Site 1 is regenerated on the opposite strand allowing Cas to nick the opposite strand, further allowing DNA polymerase reentry and the new nick site.
  • DNA polymerase extends from the new nick site, it recreates the Cas cut sites, thus allowing Cas to continue nicking the target duplex.
  • Double-stranded gDNA containing regenerated cut sites and target sequence is produced. Linear amplification of ssDNA target sequence termed Amplicon 2 is also produced.
  • Cas9n is used to create a nick at a target location. In some embodiments of methods described herein, Cas9 is used to create a blunt end at a target location. In some embodiments of methods described herein, Cas12a is used to create a DSB with a staggered 5′ overhang.
  • P1 comprises modified primer sequence complementary to the 3’ end of Amplicon 1, a promoter sequence, a Cas Nick Site 1 sequence, and a PAM1-regeneration sequence.
  • the promoter sequence is a T7 RNA polymerase promoter sequence (e.g., 5' – TAATACGACTCACTATAG – 3'.
  • the promoter sequence is a T3 RNA polymerase promoter sequence (e.g., 5′ AATTAACCCTCACTAAAG 3′). In some embodiments the promoter sequence is an SP6 RNA polymerase promoter sequence (e.g., 5 ⁇ -ATTTAGGTGACACTATAG - 3 ⁇ ). In some embodiments, DNA polymerase extends the 3’ ends of both Amplicon 1 and modified primer 1. The newly synthesized dsDNA product now contains a promoter sequence, PAM1, and Cas Nick Site 1, but on the opposite strand. In some embodiments, the RNA polymerase that corresponds to the promoter sequence is added to the reaction and binds to the promoter eliciting RNA transcription.
  • RNA polymerase synthesizes an ssRNA copy of Amplicon 1 termed Ramplicon 1.
  • Primer 2 contains a portion of Nick Site 1 and the complement for the PAM sequence for Cas on the 5’end and long complementary region of Amplicon 2 on the 3’ end. DNA polymerase extends both 3’ ends. Cas Nick Site 1 is regenerated on the opposite strand. Cas continues to nick the opposite strand making it a site for DNA polymerase reentry. As DNA polymerase extends, ssDNA product Amplicon 3 is continually displaced. This results in linear amplification of Amplicon 3.
  • CINA Strategy 2 [00088]
  • the methods introduce two DSBs and two nicks in proximity near each other in a linear portion of nucleic acid.
  • the two DSBs and two nicks are used as part of CINA Strategy 2.
  • One or more DSBs and one or more nicks may be introduced at the same time or sequentially.
  • the one or more DSBs and one or more nicks made sequentially may be made using sequential steps.
  • a Cas protein or functional fragment thereof creates one or two staggered DSBs.
  • the one or two staggered DSB are created by targeting a specific region of nucleic acid with Cas12a.
  • targeted Cas protein introduction of two staggered nicks equates to one DSB with one or two sticky ends.
  • a Cas protein or functional fragment thereof creates one or two blunt DSB.
  • the one or two blunt DSBs are created by targeting a specific region of nucleic acid with Cas9n.
  • DSBs The introduction of two DSBs allows a portion of nucleic acid surrounding the target site to be excised.
  • Appropriate sgRNAs are used to target one or more Cas proteins to a target region of interest near the sites of nucleic acid amplification.
  • a nick is created at a location in close linear proximity to a DSB.
  • a single Cas nickase can be targeted to multiple locations near a nucleic acid target of interest to create both a DSB1 site, a DSB2 site, a Nick Site 1, and a Nick Site 2.
  • two Cas proteins can be targeted to multiple locations near a nucleic acid target of interest to create both a DSB1 site, a DSB2 site, a Nick Site 1, and a Nick Site 2.
  • two or more Cas proteins can be targeted to multiple locations near a nucleic acid target of interest to create both a DSB1 site and a Nick Site 1.
  • two or more Cas proteins can be targeted to multiple locations near a nucleic acid target of interest to create both a DSB2 site and a Nick Site 2.
  • three Cas proteins can be targeted to multiple locations near a nucleic acid target of interest to create both a DSB1 site and a Nick Site 1.
  • three Cas proteins can be targeted to multiple locations near a nucleic acid target of interest to create both a DSB2 site and a Nick Site 2.
  • the staggered DSB1 or the staggered DSB2 site has a 3’ overhang.
  • the staggered DSB1 or DSB2 site has a 3’ overhang with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases overhung.
  • the staggered DSB1 or DSB2 site has a 5’ overhang.
  • the staggered DSB1 or DSB2 site has a 5’ overhang with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases overhung.
  • PAM recognition sites corresponding to the chosen Cas protein for each targeted location reside nearby the site of strand cleavage in the target.
  • Introduction of a DSB WSGR Docket No. 58557-707.601 and a nick in the target nucleic acid sequence is used to introduce a strand displacement polymerase.
  • the strand displacement polymerase (e.g., Klenow fragment [-exo]) extends the nucleotide sequence from then Nick Site 1 and Nick Site 2 each in the 3’ direction until they reach a DSB site.
  • ssDNA extended from Nick Site 1 will extend until it reaches DSB 2 .
  • the reaction continues with the regeneration of Nick Site 1 and subsequence Cas targeted nicking at Nick Site 1. This produces linear amplification of ssDNA1.
  • ssDNA extended from Nick Site 2 will extend until it reaches DSB1.
  • the reaction continues with the regeneration of Nick Site 2 and subsequence Cas targeted nicking at Nick Site 2. This produces linear amplification of ssDNA 2 .
  • Both of ssDNA 1 and ssDNA 2 contain the target amplification sequence.
  • Primer 1 (P1) binds to ssDNA2.
  • ssDNA1 and ssDNA2 are complementary in the middle portion and may form a staggered duplex through base pairing hybridization.
  • P1 binds ssDNA2 near the 3’ end and DNA polymerase extends in the 3’ direction using ssDNA2 as a template and displacing ssDNA3. Cas continues to nick regenerated Cas Nick Site 1 allowing for DNA polymerase to reenter at the newly synthesized and subsequently nicked Nick Site1.
  • CINA Strategy 2 proceeds to Part C to generate a dsDNA target.
  • ssDNA 3 and ssDNA 2 are complementary in a middle portion and may form a staggered duplex through base pairing.
  • Primer 2 contains sequence complementary to the 3’ end of ssDNA1 and bind to the region through base pairing. Following binding of P2 to ssDNA3, DNA polymerase extends in the 3’ direction using ssDNA 3 as a template. This extension starting at the 3’ end of P2 displaces ssDNA2 and regenerates Nick Site 2 for a subsequent round of nicking. Cas protein targeted to Nick Site 2 cleaves that site allowing DNA polymerase to enter at the newly synthesized nick site and extend to the 3’ end using ssDNA 3 as template. This displaces ssDNA 4 and regenerates the dsDNA template for the next round of nicking.
  • ds- gDNA containing regenerated PAM2, Nick Site 2, and target sequence is produced.
  • Linear amplification of ssDNA 4 occurs.
  • ssDNA 3 and ssDNA 4 are complementary portions of target sequence.
  • Linear amplification of both ssDNA 3 and ssDNA 4 allows for hybridization and dsDNA target sequence termed dsDNA4.
  • dsDNA4 may be detected and quantified using standard methods of dsDNA detection and quantification (e.g., ethidium bromide staining WSGR Docket No. 58557-707.601 following agarose gel electrophoresis, SYBR TM Green staining in real-time or following cessation of amplification reaction).
  • DBS within or near a target nucleotide region of interest can be generated by a number of means.
  • RNA-guided targeted cleavage by a Cas protein is one means.
  • one or more restriction enzymes is used in a reaction mix to create a DBS.
  • digestion of a target region and/or target amplicon with a restriction enzyme produces a blunt-ended DBS.
  • digestion of a target region and/or target amplicon with a restriction enzyme produces sticky-ended DBS.
  • digestion of a target region and/or target amplicon with the restriction enzyme Bmr1 produces a blunt-ended DBS.
  • treatment of a target sequence with a restriction enzyme digests the nucleic acid target dsDNA which a Cas nickase now more easily targets.
  • a double strand break (DSB) and a single strand break (nick) near each other are induced at the same time or using sequential steps, using a single Cas enzyme and appropriate gRNAs that can create a DSB on one section of the target sequence by creating 2 staggered nicks on complementary strands and a single nick on another section of the target sequence.
  • ssDNA can be excised. Any number of restriction endonuclease or nicking endonuclease known in the art may be selected to create a targeted nick or a targeted DSB in or adjacent to the target region of interest for amplification.
  • the targeted nicking or targeted DSB creation improves CINA amplification efficiency by further isolation a template nucleotide sequence for amplification from neighboring nucleotide sequence within a contiguous region of nucleic acid duplex.
  • Exemplary restriction endonuclease and nicking endonucleases are listed in Table 1. Table 1: Test restriction endonuclease and nicking endonucleases WSGR Docket No. 58557-707.601 [00092]
  • Nucleic acid targets [00093] Any cell type or tissue may be utilized to obtain nucleic acid samples for use in the targeted amplification methods described herein.
  • a target nucleic acid sample may comprise a sample from one or more sources of biological material.
  • samples may be pools and assayed together. In some embodiments, samples may be assayed individually. In some embodiments, samples may have nucleic acid purified for in vitro reaction. In some embodiments, samples may have nucleic acid present in the sample but not separate from other sample components.
  • a target nucleic acid sequence is a DNA sequence. In some embodiments, a target nucleic acid sequence is an RNA sequence. In some embodiments, the nucleic acid sequence or sequences to which a Cas protein is targeted are a DNA sequence or DNA sequences. In some embodiments, the DNA sequence or DNA sequences correspond to a genomic DNA sequence at a given location within a genome or genomic DNA sequences at given locations within a genome.
  • the DNA or RNA sample is obtained from a neoplasia, a tumor or a bodily fluid, e.g., blood, obtained by known techniques (e.g., venipuncture) or saliva.
  • nucleic acid amplification can be performed on dry samples (e.g., hair or skin).
  • CINA protocols may be used to amplify one or more DNA sequences of derived from a plasmid.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of one or more bacterial species.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of one or more Archaea species.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of one or more eukaryotic species.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of one or more plant species.
  • the one or more plant species is Arabidopsis thaliana, Hordeum vulgare, Arabis alpina, Capsella rubella, Zea mays, Physaria filiformis, Nicotiana WSGR Docket No.
  • CINA protocols may be used to amplify one or more DNA sequences derived from a viral genome.
  • the viral genome is from influenza virus A, influenza virus B, highly pathogenic avian influenza (HPAI), SARS-CoV-2, African swine fever virus (ASFV), White spot syndrome (WSS), respiratory syncytial virus A (RSV A), RSV B, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Legionella micdadei, Bordetella pertussis, Staphylococcus aureus, and/or Streptococcus pneumoniae.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of one or more fungal genus and/or species.
  • the one or more fungal genus and/or species may be Candida albicans, Aspergillus, Cladosporium herbarum, Saccharomyces cerevisiae, Penicillium, Acremoium, Alternaria, Curvularia, and/or Fusarium.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of one or more animal species.
  • the one or more animal species may be selected from the phyla Porifera, Cnidaria, Platyhelminthes, Nematoda, Mollusca, Annelida, Echinodermata, Arthropoda, or Chordata.
  • the arthropod animal species belongs to a genus of mosquito.
  • the mosquito genus is Aedes, Anopheles, Culex.
  • the mosquito species is Aedes aegypti or Aedes albopictus.
  • the Chordata species belongs to a class of fish, amphibians, reptiles, birds, or mammals.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of a human subject.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of a mouse subject.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of a rat subject.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of a canine subject.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of a feline subject.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of a porcine subject.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of a bovine subject.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of an equine subject.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of a goat subject.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of a camel subject.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of a llama subject.
  • CINA protocols may be used to WSGR Docket No. 58557-707.601 amplify one or more DNA sequences derived from genomic DNA of a sheep subject.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of a chicken subject.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of a duck subject.
  • CINA protocols may be used to amplify one or more DNA sequences derived from genomic DNA of a turkey subject.
  • ss amplicons of a given size are able to be efficiently and specifically amplified from a sample.
  • one or more ds amplicons of a given size are able to be efficiently and specifically amplified from a sample.
  • the size of the ss amplicons or the one or more ds amplicons is between about 50-5000 nucleotides in length. In some embodiments, the size of the ss amplicons or the one or more ds amplicons is between about 70- 4000 nucleotides in length.
  • the size of the ss amplicons or the one or more ds amplicons is between about 70-2000 nucleotides in length. In some embodiments, the size of the ss amplicons or the one or more ds amplicons is between about 70-1000 nucleotides in length. In some embodiments, the size of the ss amplicons or the one or more ds amplicons is between about 70-500 nucleotides in length. In some embodiments, the size of the ss amplicons or the one or more ds amplicons is between about 70-300 nucleotides in length.
  • the size of the ss amplicons or the one or more ds amplicons is between about 70- 250 nucleotides in length. In some embodiments, the size of the ss amplicons or the one or more ds amplicons is between about 80-200 nucleotides in length. In some embodiments, the size of the ss amplicons or the one or more ds amplicons is between about 90-180 nucleotides in length.
  • the size of the ss amplicons or the one or more ds amplicons is greater than 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 nucleotides in length.
  • the size of the ss amplicons or the one or more ds amplicons is less than 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nucleotides in length.
  • the target region to be amplified is from 20 to 500 bases in length. In other embodiments, the target region is from 20 to 400 bases in length. In some embodiments, the target region is from 20 to 300 bases in length.
  • the target region is from 20 to 200 bases in length. In some embodiments, the target region is from 20 to 100 bases in length. In some embodiments, the target region is from 20 to 50 bases in length. In some embodiments, the target region is from 20 to 25 bases in length. In some embodiments, the target region is from 25 to 500 bases in length. In some embodiments, the target region is from 25 to 400 bases in length. In some WSGR Docket No. 58557-707.601 embodiments, the target region is from 25 to 300 bases in length. In some embodiments, the target region is from 25 to 200 bases in length. In some embodiments, the target region is from 25 to 100 bases in length. In some embodiments, the target region is from 25 to 50 bases in length.
  • the target region is from 50 to 500 bases in length. In some embodiments, the target region is from 50 to 400 bases in length. In some embodiments, the target region is from 50 to 300 bases in length. In some embodiments, the target region is from 50 to 200 bases in length. In some embodiments, the target region is from 50 to 100 bases in length. In some embodiments, the single-stranded amplicons are from 20 to 500 bases in length. In other embodiments, the single-stranded amplicons are from 20 to 400 bases in length. In some embodiments, the single-stranded amplicons are from 20 to 300 bases in length. In some embodiments, the single-stranded amplicons are from 20 to 200 bases in length.
  • the single-stranded amplicons are from 20 to 100 bases in length. In some embodiments, the single-stranded amplicons are from 20 to 50 bases in length. In some embodiments, the single-stranded amplicons are from 20 to 25 bases in length. In some embodiments, the single-stranded amplicons are from 25 to 500 bases in length. In some embodiments, the single-stranded amplicons are from 25 to 400 bases in length. In some embodiments, the single-stranded amplicons are from 25 to 300 bases in length. In some embodiments, the single-stranded amplicons are from 25 to 200 bases in length. In some embodiments, the single-stranded amplicons are from 25 to 100 bases in length.
  • the single-stranded amplicons are from 25 to 50 bases in length. In some embodiments, the single-stranded amplicons are from 50 to 500 bases in length. In some embodiments, the single-stranded amplicons are from 50 to 400 bases in length. In some embodiments, the single-stranded amplicons are from 50 to 300 bases in length. In some embodiments, the single-stranded amplicons are from 50 to 200 bases in length. In some embodiments, the single-stranded amplicons are from 50 to 100 bases in length. In some embodiments of methods described herein, primer systems are used with CINA reaction reagents to facilitate amplification of target amplicons. In some embodiments, two or more target region amplicons are produced.
  • Detecting CINA Reaction Product [00097] In some methods described herein, one or more ssDNA amplicons is detected by Lateral Flow Assay (LFA). In some embodiments, LFA with FAM-labelled and/or biotin- labelled probes is used in the method to detect target ssDNA amplicon. In some embodiments, WSGR Docket No.
  • LFA with FAM-labelled and/or biotin-labelled primers is used in the method to detect target ssDNA amplicon.
  • two independently labelled probes bind to unlabeled ssDNA 1.
  • a FAM probe and a Biotinylated probe bind to different regions of unlabeled ssDNA. The probes can then bind to FITC antibody and streptavidin, respectively. This binding permits detection of unlabeled amplicon by LFA.
  • Reaction Temperature [00099]
  • a CINA method is used to conduct a nucleic acid amplification reaction at about an ambient temperature.
  • a CINA method is used to conduct a nucleic acid amplification reaction at an ambient temperature.
  • Ambient temperature may be about 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C,
  • a CINA method is used to conduct a nucleic acid amplification reaction under conditions in which the ambient temperature changes during the duration of the amplification reaction by less than about 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3.5°C, 3°C, 2.5°C, 2°C, 1.75°C, 1.5°C, 1.25°C, 1.0°C, 0.9°C, 0.8°C, 0.7°C, 0.6°C, 0.5°C, 0.4°C, 0.3°C, 0.25°C, 0.2°C, 0.15°C, 0.1°C, 0.05°C, 0.04°C, 0.03°C, 0.02°C, or 0.01°C.
  • a CINA method is used to conduct a nucleic acid amplification reaction under near isothermal reaction conditions. In some embodiments, a CINA method is used to conduct a nucleic acid amplification reaction under isothermal reaction conditions. In some embodiments, a CINA method is used to conduct a nucleic acid amplification reaction in a temperature range between 25°C - 65°C. In some embodiments, a CINA method is used to conduct a nucleic acid amplification reaction in a temperature range between 25°C - 45°C. In some embodiments, a CINA method is used to conduct a nucleic acid amplification reaction in a temperature range between 25°C - 37°C.
  • a CINA method is used to conduct a nucleic acid amplification reaction in a temperature range between 37°C - 65°C. In some embodiments, a CINA method is used to conduct a nucleic acid amplification reaction in a temperature range between 37°C - 45°C. In some embodiments, a CINA method is used to conduct a nucleic acid amplification reaction in a temperature at least about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C.
  • a CINA method is used to conduct a nucleic acid amplification reaction in a temperature at least about 25°C. In some embodiments, a CINA method is used to conduct a nucleic acid amplification reaction in a temperature at least about 30°C. In some embodiments, a CINA method is used to conduct a nucleic acid amplification reaction in a WSGR Docket No. 58557-707.601 temperature at least about 37°C. In some embodiments, a CINA method is used to conduct a nucleic acid amplification reaction in a temperature less than about 65°C. In some embodiments, a CINA method is used to conduct a nucleic acid amplification reaction in a temperature less than about 60°C.
  • a CINA method is used to conduct a nucleic acid amplification reaction in a temperature less than about 55°C. In some embodiments, a CINA method is used to conduct a nucleic acid amplification reaction in a temperature less than about 50°C. In some embodiments, a CINA method is used to conduct a nucleic acid amplification reaction in a temperature less than about 45°C. In some embodiments, a CINA method is used to conduct a nucleic acid amplification reaction in a temperature less than about 42°C. In some embodiments, a CINA method is used to conduct a nucleic acid amplification reaction in a temperature less than about 37°C.
  • a CINA method is used to conduct a nucleic acid amplification reaction in a temperature about 25°C. In some embodiments, a CINA method is used to conduct a nucleic acid amplification reaction in a temperature about 37°C. In some embodiments, a CINA method is used to conduct a nucleic acid amplification reaction in a temperature about 45°C. In some embodiments, an optimized temperature results in efficient targeting nicking of one or more Nick-Sites near the target amplification sequence. In this instance, near the target amplification sequence generally refers to a location within about 300 bp of a nucleotide region to be amplified.
  • CINA Reaction Optimization [000101]
  • a method of isothermal nucleic acid amplification described herein is optimized to produce more efficient target sequence amplification.
  • the method is optimized to reduce off-target sequence amplification.
  • the method is optimized to increase specific production of one or more target amplicons.
  • the reaction buffer is optimized to improve activity of the DNA polymerase.
  • a Cas9n NEB buffer r3.1 is used.
  • a Cas9n NEB buffer r3.1 is used Bst 3.0 Isothermal amplification buffer (IAB2) is used.
  • IAB2 Isothermal amplification buffer
  • amplification is more efficient in buffer IAB2 than in buffer r3.1.
  • the ratio of Cas9n:sgRNA is optimized.
  • the Cas9n:sgRNA ratio is between about 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:01.
  • the Cas9n:sgRNA molar ratio is about 1:1. In some embodiments, the Cas9n:sgRNA molar ratio is about 1:1.5.
  • the Cas9n:sgRNA molar ratio is about 1:2. In some embodiments, the Cas9n:sgRNA concentration is optimized. In some embodiments, the WSGR Docket No. 58557-707.601 Cas9n:sgRNA concentration is about 100:100 nM. In some embodiments, the Cas9n:sgRNA concentration is about 100:150 nM. In some embodiments, the Cas9n:sgRNA concentration is about 100:200 nM. In some embodiments, the Cas9n:sgRNA concentration is about 10:10 nM. In some embodiments, the Cas9n:sgRNA concentration is about 30:30 nM.
  • the Cas9n:sgRNA concentration is about 100:100 nM. In some embodiments, the Cas9n:sgRNA concentration is about 200:200 nM. In some embodiments, the Cas9n:sgRNA concentration is about 300:300 nM. In some embodiments, the concentration of template DNA is optimized. In some embodiments, the concentration of template DNA is about 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 17, 19, 20, 22, 24, 25, 27, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nM.
  • the concentration of template DNA is about 1 nM. In some embodiments, the concentration of template DNA is about 1 nM. In some embodiments, the concentration of template DNA is about 3 nM, In some embodiments, the concentration of template DNA is about 10 nM. In some embodiments, the concentration of template DNA is about 20 nM. In some embodiments, the concentration of template DNA is about 30 nM. In some embodiments, the Cas9n:sgRNA:DNA ratio is optimized. In some embodiments, the Cas9n:sgRNA:DNA ratio is 30:30:3. In some embodiments, the Cas9n:sgRNA:DNA ratio is about 12.5:25:3.
  • the Cas9n:sgRNA:DNA ratio is 200:100:10.
  • the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
  • each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and/or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.
  • WSGR Docket No. 58557-707.601 [000106]
  • “or” may refer to “and”, “or,” or “and/or” and may be used both exclusively and inclusively.
  • the term “A or B” may refer to “A or B”, “A but not B”, “B but not A”, and “A and B”.
  • any systems, methods, software, and platforms described herein are modular. Accordingly, terms such as “first” and “second” do not necessarily imply priority, order of importance, or order of acts.
  • the term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and the number or numerical range may vary from, for example, from 1% to 15% of the stated number or numerical range. In examples, the term “about” refers to ⁇ 10% of a stated number or value.
  • the terms “increased”, “increasing”, or “increase” are used herein to generally mean an increase by a statically significant amount.
  • the terms “increased,” or “increase,” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, standard, or control.
  • “increase” include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.
  • the terms “decreased”, “decreasing”, or “decrease” are used herein generally to mean a decrease by a statistically significant amount.
  • “decreased” or “decrease” means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level.
  • a marker or symptom by these terms is meant a statistically significant decrease in such level.
  • expression, activity, or level of a reporter gene can be measured, and sgRNA: nucleases targeting a gene sequence can be assayed for their ability to reduce the expression, activity, or level of the gene sequence expression.
  • a cell can be transfected with an expression cassette encoding a green fluorescent protein under the control of a constitutive promoter. The fluorescence intensity can be measured and compared to the intensity of the cell after transfection with Cas9 and candidate sgRNAs to identify optimized sgRNAs.
  • editing enzyme means an enzyme isolated from a eukaryote or prokaryotic cell that is capable of catalyzing a reaction between one or a plurality of nucleotides to generate a nucleic acid sequence or a replicate of a template nucleic acid sequence.
  • the editing enzyme is a DNA editing enzyme, such as DNA polymerase.
  • editing enzymes include fusion proteins with a nucleic acid-editing domain, such as those found in USPN 11,124,78, of which the editing enzymes described therein are incorporated by reference in its entirety.
  • a functional fragment means any portion or fragment of a polypeptide or nucleic acid sequence from which the respective full-length polypeptide or nucleic acid relates that is of a sufficient length and has a sufficient structure to confer a biological affect that is similar or substantially similar to the full-length polypeptide or nucleic acid upon which the fragment is based.
  • a functional fragment refers to a fragment of an enzyme, such as an editing enzyme, a polymerase, or a Cas protein such as Cas 9, Cas12, or Cas12a, disclosed herein that comprises at least about 75% sequence identity to the wild-type protein.
  • the functional nature of the fragment is to bind or associate a disclosed nucleic acid target sequence, and in some embodiments, cut the DNA target sequence.
  • a functional fragment is a portion of a full-length or wild-type nucleic acid sequence that encodes any one of the nucleic acid sequences disclosed herein, and said portion encodes a polypeptide of a certain length and/or structure that is less than full-length but encodes a domain that still biologically functional as compared to the full-length or wild-type protein.
  • the functional fragment may have a reduced biological activity, about equivalent biological activity, or an enhanced biological activity as compared to the wild- type or full-length polypeptide sequence upon which the fragment is based.
  • the functional fragment is derived from the sequence of an organism, such as a human.
  • the functional fragment may retain about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, or about 90% sequence identity to the wild-type or given sequence upon which the sequence is derived.
  • the functional fragment may retain about 85%, about 80%, about 75%, WSGR Docket No. 58557-707.601 about 70%, about 65%, or about 60% sequence homology to the wild-type sequence upon which the sequence is derived.
  • the enzymes with mutations (such as the Cas9 enzyme) or any functional fragments thereof described herein are intended to include amino acid sequences comprising polypeptides bearing one or more insertions, deletions, or substitutions, or any combination thereof, of amino acid residues as well as modifications other than insertions, deletions, or substitutions of amino acid residues, such as but not limited to conservative amino acid substitutions.
  • “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self- hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR or initiation of the activity of a polymerase, or the cleavage of a polynucleotide by an enzyme.
  • a sequence capable of hybridizing with a given sequence is referred to as the "complement" of the given sequence.
  • Nucleotide base pairing occurs by hybridization. [000116] "Nucleoside" means a nucleobase linked to a sugar moiety.
  • Nucleotide means a nucleoside having a phosphate group covalently linked to the sugar portion of a nucleoside.
  • the nucleotide is characterized as being modified if the 3' phosphate group is covalently linked to a contiguous nucleotide by any linkage other than a phosphodiester bond.
  • nucleic acid molecules e.g., cDNA or genomic DNA
  • RNA molecules e.g., mRNA
  • analogs of the DNA or RNA generated using nucleotide analogs e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs
  • hybrids thereof e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs
  • the nucleic acid molecule can be single-stranded or double-stranded.
  • the nucleic acid molecules of the disclosure comprise a contiguous open reading frame encoding an antibody, or a fragment thereof, as described herein.
  • Nucleic acid or “oligonucleotide” or “polynucleotide” as used herein may mean at least two nucleotides covalently linked together.
  • the depiction of a single strand also defines the sequence of the complementary strand.
  • a nucleic acid also encompasses the complementary strand of a depicted single strand.
  • Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid.
  • a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.
  • a WSGR Docket No. 58557-707.601 single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions.
  • nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.
  • Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequence.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine
  • Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.
  • dNTP deoxynucleotide triphosphate.
  • nucleotide triphospahtes are deoxyribose nucleotide triphopshates or ribose nucleotide triphosphates.
  • Strategy 1 for CRISPR-assisted isothermal nucleotide amplification (CINA) [000122]
  • CINA CRISPR-assisted isothermal nucleotide amplification
  • a nucleotide amplification strategy is demonstrated using one or more CRISPR Cas enzymes and single guide RNAs (sgRNAs) to target the one or more Cas enzymes to target loci.
  • sgRNAs single guide RNAs
  • one targeted nick and one targeted DSB are produced near each WSGR Docket No. 58557-707.601 other in a selected target region of genomic DNA.
  • the one targeted nick and one targeted DSB can be introduced at the same time or using sequential steps.
  • an sgRNA-guided Cas enzyme creates either a blunt DSB or a staggered DSB in a target region of gDNA and a different sgRNA-guided Cas enzyme creates a nick.
  • the Cas enzyme Cas12a creates the staggered DSB.
  • the Cas effector Cas12a is RNA- guided endonuclease.
  • the different Cas enzyme, Cas9n D10A creates the nick.
  • the Cas effector Cas9n D10A is an RNA-guided nicking endonuclease (nickase).
  • Cas9n D10A is also referred to as Spy Cas9 nickase (Cas9n).
  • Streptococcus pyogenes Cas9 also known as wildtype Cas9, (GenBank accession no. NZ_LS483338, GeneID: 69900934), may be used instead of Cas12a to create a blunt DSB.
  • the DSB either staggered or blunt
  • the nick are utilized to introduce a strand displacement DNA polymerase, in this case Klenow Fragment, to carry out extension and linear amplification.
  • Cutting properties are determined by the choice of Cas effector.
  • the CRISPR-Cas systems in this example are made of an sgRNA and a Cas nuclease, which together form a ribonucleoprotein complex. Cut sites for the Cas effectors are determined by the locations of PAM sequences within the target region of gDNA. The presence of PAM sequences in the gDNA is requires for a sgRNA to bind to a target sequence. The Cas effector binds to its target sequence in the presence of PAM, o the non-targeted DNA strand.
  • the sgRNA comprises a short crispr RNA (crRNA) fused to a scaffold trans-activating CRISPR (tracr)RNA sequence.
  • the crRNA portion of the sgRNA comprise nucleotide sequence complementary to the target DNA, thus targeting the ribonucleoprotein complex to a specific location within genome based on linear DNA sequence of the target DNA.
  • the tracrRNA portion of the sgRNA serves as a binding scaffold for the Cas nuclease.
  • the Cas effector can cut DNA in the targeted locus according to the locations of adjacent PAM sequence.
  • sgRNA sequences are selected based on targeting locations in gDNA and with additional considerations for efficiency of genomic targeting. These additional considerations include selecting a GC content of the sgRNA to be between 40-80%. A higher GC content yields a more stable sgRNA.
  • Exemplary targeted Cas proteins for use in Strategy 1 include Spy Cas9 nickase (Cas9n) with a guide RNA target Nick-Site 1 and Seq 1 Cas9 or Lba Cas12a with a guide RNA targeting DSB 1.
  • a single Cas enzyme guided by appropriate sgRNAs creates a staggered DSB on one section of the target sequence by creating two staggered nicks on complementary strands in a target region of gDNA and a single nick on WSGR Docket No. 58557-707.601 another section of the target sequence.
  • Cas9n D10A is the Cas effector creating the two staggered nicks and the single nick and is targeted to the chosen locations by selecting sgRNA sequences complementary to the particular target location gDNA sequences.
  • FIG.3 shows two images of agarose gel products (1.5% agarose gel, run at 160V for 50 min) analyzed by electrophoresis and nucleic acid labeling.
  • Targeting pUC19 plasmid, 2 different Cas enzymes were demonstrated to work together to create separate nicks and DSBs.
  • Open circle plasmid is shown in FIG.3 following gRNA targeting of Cas9n.
  • Purified plasmid exists primarily in a supercoiled (SC) state.
  • SC supercoiled
  • OC open circle
  • Cas12a was used to with a gRNA targeting sequence to target a site in pUC19 plasmid to produce a double strand break (DSB).
  • DSB double strand break
  • Production of a DSB was demonstrated in FIG.3 as shown to create a linear (LIN) plasmid.
  • Cas9 nickase (Cas9n) with specific sgRNA nicks plasmid was shown to produce open circle plasmid.
  • Cas 12a with specific gRNA makes double strand breaks (DSBs) in plasmid was shown to create linearized plasmid. All lanes in FIG.3 containing Cas12a DSBs and Cas9 nickase produce both nicked (OC) and linearized (LIN) species. Both nicked and linear species are present in each sample containing Cas12a and Cas9n, indicating simultaneous nicking and DSBs are successful. This is successful demonstration that two different Cas enzymes can work together to create separate nicks and DSBs. [000125] CINA Strategy 1 and CINA Strategy 2 described herein both involve strand displacing DNA polymerase entering a 3’ end of nicked DNA and displace ssDNA amplicons.
  • a strand displacing DNA polymerase can dislodge the Cas-complex, extending the 3’ end of nicked DNA, regenerate a nick site and displace ssDNA.
  • three DNA polymerases were tested (Klenow Fragment, Bst 2.0, and Phi Polymerase). Open circle (OC) pUC19 plasmid was obtained and reacted separately with each of these DNA polymerases in the presence of dNTPs and appropriate buffer.
  • the DNA polymerase selected (e.g., Klenow Fragment lacking 5’ to 3’ or 3’ to 5’ exonuclease activity [-exo]) extends the 3’ end of the nicked strand until it reaches to the location of the DSB. By doing so, the DNA polymerase recreates Nick- Site 1, allowing the Cas effector that targeted Nick-Site 1 to continue nicking the target duplex. This allows the DNA polymerase to extend the 3’ end of the newly synthesized nicks and simultaneously displacing the ssDNA target sequence. Cas9n D10A nicks as Nick-Site 1 and Cas12a creates a staggered DSB at cut-site 2.
  • ssDNA target termed Amplicon 1 in FIG.5.
  • the products of Part A are i) ds-gDNA containing regenerated Nick-Sites and target sequence and ii) ssDNA target sequence termed Amplicon 1 that has been subjected to linear amplification. This amplification is accomplished without the use of a primer. In some embodiments, the reaction is stopped following a period of time of Part A.
  • Linearly amplified ssDNA Amplicon 1 can be detected and quantified by probe using any number of standard detection assays (e.g., a protocol according to use of the QuantiFluor® ssDNA System; Promega, Madison, WI).
  • Step 4 The reaction can be stopped here at Part A, and Amplicon 1 can be detected by probe. To achieve exponential amplification, Part 2 and Part 3 need to be added.
  • Part B 1 of Strategy 1 for CINA Primer 1 (P1) is included in the reaction mixture.
  • P1 contains i) cut-site 1 sequence and ii) sequence complementary to the PAM WSGR Docket No. 58557-707.601 sequence for the Cas effector chosen to target the 5’ end of the target sequence, and iii) a complementary region to Amplicon 1 on the 3’ end.
  • the ssDNA Amplicon 1 from Part A is now free to bind to P1.
  • the DNA polymerase e.g., Klenow Fragments [-exo], Bst 2.0, Phi polymerase, or a combination thereof
  • the DNA polymerase extends both 3’ ends thereby regenerating the sequence of Nick-Site 1 on the reverse strand.
  • the Cas effector nickase e.g., Cas9n D10A
  • the extension recreates the Cas cut-sites, allowing Cas effector to continue nicking the target duplex.
  • the repeating of primer binding and extension of Part B linearly amplifies ssDNA Amplicon 2.
  • the products derived from Part B are i) ds-gDNA containing regenerated cut-sites and target sequence and ii) linear amplification of ssDNA target sequence termed Amplicon 2.
  • Bst 2.0, Phi polymerase, or a combination of DNA polymerases may be used.
  • Step 3) Steps 1 and 2 of Part B are repeated for the opposite strand which produces linear amplification of ssDNA Amplicon 2.
  • an alternative format termed Part B 2 may be used to generate ssRNA copies of Amplicon 1.
  • Amplicon 1 and Amplicon 2 are complementary and may be able to bind to each other and stop the CINA reaction from continuing to Part C.
  • Part B2 modified primer 1 (MP1) is used in the reaction and can bind to the 3’ end of Amplicon 1.
  • MP1 comprises sequence complementary to the 3’ end of Amplicon 1, a promoter sequence (e.g., T7, T3, or SP6), a Cas effector Nick-Site 1, and a PAM-regeneration sequence.
  • DNA polymerase (e.g., Klenow Fragments [-exo], Bst 2.0, Phi polymerase, or a combination thereof) extends the 3’ ends of both Amplicon 1 and MP1 creating newly synthesized dsDNA product now containing the promoter sequence, PAM sequence, and Cas Nick-Site 1 sequence on the reverse strand. This will ensure that dsDNA products will have the promoter sites.
  • RNA polymerase in the reaction mixture binds to the promoter sequence and transcribes an ssRNA copy of Amplicon 1, termed Ramplicon 1.
  • the RNA polymerase used corresponds to the selected promoter sequence (e.g., T7-dependent RNA polymerase for T7 promoter sequence).
  • Ramplicon 1 can be detected and quantified by probe using any number of standard detection assays (e.g., in situ RNA hybridization).
  • WSGR Docket No. 58557-707.601 As shown in FIG.8, Part C of Strategy 1 for CINA includes Primer 2 (P2) containing part of Nick-Site 1 and complementary sequence for the PAM sequence for Cas on the 5’ end and long complementary region to Amplicon 2 on the 3’ end.
  • the ssDNA Amplicon 2 from Part B is now free to bind to P2.
  • DNA polymerase e.g., Klenow Fragments [-exo]
  • DNA polymerase extends both 3’ ends regenerating the Cas effector Nick-Site 1 on the forwards strand.
  • Products from Part C include i) ds-gDNA containing regenerated cut-sites and target sequence, and ii) linear amplification of ssDNA target sequence termed Amplicon 3.
  • nicking and extension steps described in Part are repeated, linearly amplifying ssDNA Amplicon 3, which is identical to Amplicon 1. This combined effect produces exponential amplification of target ssDNA strands that can be detected and quantified by a probe.
  • Steps in CINA Strategy 1, Part C, as seen in FIG.8 • Step 1) Primer Binding: The ssDNA Amplicon 2 from Part B is now free to bind • Step 2) Extension: Klenow Fragment extends P2 and recreates Cas Nick-Site 1. In this instance, Cas Nick-Site 1 is recreated on the original strand. Alternatively, Bst 2.0, Phi polymerase, or a combination of DNA polymerases may be used.
  • Step 3) Step 1 and Step 2 of Part A are repeated, producing linear amplification of ssDNA Amplicon 3, which is identical to ssDNA Amplicon 1.
  • ssDNA Amplicon 1 and ssDNA Amplicon 3 leads to exponential amplification of target sequence. This target sequence can then be detected by a probe.
  • strategy 1and 2 for CINA the following assay reagents and design parameters are used to amplify pUC19 plasmid DNA.
  • Table 2 lists primer sequence for DNA target region binding, crRNA sequences for DNA targeting, and transcribed RNA sequences for sgRNA molecules.
  • Table 3 lists pairs of DNA targeting sequence names for sgRNA or crRNA and corresponding transcribed RNA sequence names. Table 2: Primer, crRNA, and transcribed RNA sequences WSGR Docket No.
  • LbCas12a is targeted to cleave DNA at its target location to create staggered nicks on the forward and reverse strands to produce DSB1.
  • Cas9n D10A is targeted upstream to create Nick Site 1.
  • Klenow fragment deficient in exonuclease activity extends the 3’ end of the nicked strand until it reaches DSB1. While extending the 3’ end of the nicks, Klenow fragment (exo-) simultaneously displaces the ssDNA target sequence and regenerates Nick Site 1. This allows targeted Cas9n D10A to continue nicking the target duplex. This produces linear amplification of Amplicon 1 shown in FIG.10.
  • Amplicon 1 in the form of ssDNA can now be detected via probe.
  • Primer 1 as shown in FIG.10 contains sequence complementary to i) sgRNA 1 dsDNA target, ii) PAM 1 sgRNA1, iii) the 5’ end of Amplicon 1, and iv) the Cas9n Nick Site.
  • Primer 1 anneals near the 3’ end of Amplicon 1.
  • Klenow fragment (-exo) extends the 3’ end of Amplicon 1 to complement the remainder of the sequence of Primer 1 and extends the 3’ from Primer 1.
  • Nick Site 1 is recreated on the opposite strand.
  • FIG.11 shows the product of dsDNA1 which is generated from Primer 1 extension via Amplicon 1 and a map of Amplicon 2 with locations of the regenerated sgRNA target site, Nick Site on the bottom strand, and the NGG PAM sequence.
  • Primer 2 as shown in FIG.12 contains sequence i) complementary to the 3’ of Amplicon 2, ii) regenerated Nick Site 1, and iii) regenerated PAM 1 for sgRNA 1 Cas9n nick site.
  • Step 1 and 2 of Part A are repeated, linear amplification of ssDNA Amplicon 1 and ssDNA Amplicon 3 produces exponential amplification of the target sequence.
  • Amplicon 1/Amplicon 3 and/or Amplicon 2 can be detected and quantified by a probe which will bind to ssDNA harboring sequence complementarity to target regions of pUC19 DNA.
  • DSB 2v2 (Cas12a2+ gRNA targeting DSB2v2 site) 2. 502 Nick 1 (Cas9n+ sgRNA 502Nick 1 site) 3. DSB 2v2 + 502 Nick 1 4. DSB 2v2 + 502 N1 + Bst (polymerase) 5. DSB 2v2 + 502 N1 + Bst + nP1 (nicking Primer 1) 6.
  • L4 The addition of Bst polymerase displaces ssDNA 1 which is not detectable on agarose gel, resulting in positive LFA detection (L4, LFA).
  • L5 Agarose: Contains both nicked and linear species, indicating DSBs and Nicks were successfully introduced by LbCas12a and Cas9n (L5, Agarose).
  • L5 (LFA) ssDNA 2 produced from nPrimer 1 cannot bind ssDNA probes, consistent with negative results observed on LFA (L5, LFA).
  • L6 (Agarose): Contains both nicked and linear species, indicating DSBs and Nicks were successfully introduced by LbCas12a and Cas9n (L6, Agarose).
  • Amplicons are generated using both nicking & non-nicking primers for strategy 1 using the starting template from pUC19 diagrammed in FIG.23. Reaction products were analyzed by via agarose gel electrophoresis (left), via 10% Native PAGE (center), and by LFA (right) and shown in FIG.15A.
  • LFA ssDNA 1 probes Probe 1: L.5,3-FAM-ssDNA 1-pv1A Probe 2: L.5,3-Btn-ssDNA 1-pv1B [000154] Observations: [000155] L1 and L5: 502 Nick 1 and Nick B2 respectively nick the template (500 bp dsDNA 1) but is not obvious on native PAGE or agarose gels. [000156] L2: ssDNA 1 (280 b) is displaced upon addition of Bst 3.0 which is observed in native PAGE but not agarose gels and can be detected on LFA using Probes 1 and 2.
  • L3 RP-500 transforms ssDNA 1 to dsDNA 2 (280bp) as observed in both native PAGE and agarose gels and therefore cannot be detected by LFA anymore.
  • L6 ssDNA 2 (402 b) is successfully displaced upon addition of Bst 3.0 which is observed in native PAGE.
  • L4 RP-500 regenerates Nick-B2, which can then produce ssDNA 2 (402b) and ssDNA 3 (180b) which cannot be detected by LFA.
  • FIG.15B shows band quantification results and a graph of normalized relative band intensity from the gel run in FIG.15A to quantify production of ss and ds 280 base amplicons.
  • Strategy 1 for CINA the following assay reagents and design parameters are used to amplify human EMX1 gDNA.
  • Amplicon 1 (ssDNA): [000170] 5’- GACAAAGTACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAAGAAGGGCTCCCAT CACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACCTCCAAT-3’ [000171] Primer 1: [000172] 5’-CCGGAGGACAAAGTACAAACGGCATTGGAGGTGACATC-3’ [000173] Human EMX1 mRNA sequence, Amplicon 1, and Primer 1 are listed in Table 4: Table 4: Sequences for CINA Strategy 1 amplification of human EMX1 WSGR Docket No.
  • dsDNA1 product is formed by complementary binding of forward strand and reverse strand listed below.
  • Forward Strand 5’GACAAAGTACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAAGAAGGGCTCCC ATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACCTCCAATGC CGTTTGTACTTTGTCCTCCGG-3’
  • Reverse Strand (bound to forward strand to form dsDNA1) [000177] 3’CTGTTTCATGTTTGCCGTCTTCGACCTCCTCCTTCCCGGACTCAGGCTCGTCTTCTTCTTC CCGAGGGTAGTGTAGTTGGCCACCGCGTAACGGTGCTTCGTCCGGTTACCCCTCCTGTAGCTACAGTGGA GGTTACGGCAAACATGAAACAGGAGGCC-5
  • Amplicon 2 (ssDNA): [000179] 5’- GACAAAGT
  • dsDNA2 product is formed by complementary binding of forward strand and reverse strand listed below.
  • Forward strand WSGR Docket No. 58557-707.601
  • 5’ - GACAAAGTACAAACGGCATTGGAGGTGACATCGATGTCCTCCCCATTGGCCTGCTTCGTGGCAATGCGCCACCGGTT GATGTGATGGGAGCCCTTCTTCTTCTGCTCGGACTCAGGCCCTTCCTCCTCCAGCTTCTGCCGTTTGTACTTTGTCC TCCGG-3’
  • Reverse Strand (bound to forward strand to form dsDNA2): [000187] 3’- CTGTTTCATGTTTGCCGTAACCTCCACTGTAGCTACAGGAGGGGTAACCGGACGAAGCACCGTTACGCGGTGGCCAA CTACACTACCCTCGGGAAGAAGAAGACGAGCCTGAGTCCGGGAAGGAGGAGGTCGAAGACGGCAAACATGAAACA
  • Amplicon 1, Amplicon 2, and Amplicon 3 are generated using strategy described in FIG.1 - FIG.2 and FIG.5- FIG.8 and in Strategy 1-A for puc19. [000191] Following Steps 1 and 2, Amplicon 1/Amplicon 3 and/or Amplicon 2 can be detected and quantified by a probe which will bind to ssDNA harboring sequence complementarity to target regions of human EMX2 DNA. Example 2.
  • Strategy 2 for CRISPR-assisted isothermal nucleotide amplification (CINA) [000192]
  • CINA CRISPR-assisted isothermal nucleotide amplification
  • a nucleotide amplification strategy is demonstrated using one or more CRISPR Cas enzymes and single guide RNAs (sgRNAs) to target the one or more Cas enzymes to target loci.
  • Cas effector introduces two DSBs to isolate a region of gDNA comprising the target sequence.
  • DSB 1 and DSB 2 are created by sgRNA-guided Cas effector proteins targeted to Cas Cut-Site A (for DSB 1 ) and Cas Cut-Site B (for DSB 2 ).
  • Cas Cut-Sites reside adjacent to, but outside of the region comprising Cas Nick-Site 1, Cas Nick-Site 2, and the target amplification region.
  • PAM sequences enabling the endonuclease or nickase activity at the selected regions of the target sites are indicated in FIG.16.
  • DSB1 and DSB2 are created as blunt ends with the use of sgRNA-targeted wildtype Cas9 or staggered ends with the use of sgRNA-targeted Cas12a.
  • Strategy 2 also uses the generation of two distinct nicks and one primer in the amplification procedure.
  • DNA polymerase extends the 3’ ends of each nick while simultaneously displacing ssDNA1 (forward strand containing the red box), ssDNA 2 (reverse strand) and regenerating both Cas Nick-Sites and corresponding PAM sequences for each strand.
  • ssDNA 1 forward strand containing the red box
  • ssDNA 2 reverse strand
  • double-stranded gDNA template contain the regenerated Nick-Sites and target sequence is also produced.
  • ssDNA1 and ssDNA2 are complementary in a partial overlap and can form a staggered duplex.
  • Primer 1 (P1) binds to ssDNA 2 and DNA polymerase (e.g., Klenow Fragments [-exo]) extends 3’ using ssDNA2 as template. This displaces ssDNA1 and regenerates Cas Nick-Site 1 for additional rounds of nicking from Cas effector nickase. Klenow Fragments re-enter the newly synthesized nick site and displaces the top strand (ssDNA 3 ), regenerating the dsDNA template for the next round of nicking.
  • P1 binds to ssDNA 2 and DNA polymerase (e.g., Klenow Fragments [-exo]) extends 3’ using ssDNA2 as template.
  • Klenow Fragments re-enter the newly synthesized nick site and displaces the top strand (ssDNA 3 ), regenerating
  • ssDNA3 The displaced forward strand upstream of Nick-Site 1 (ssDNA3) is amplified in a linear manner and contains the target sequence (boxed in red).
  • Parts A + B of Strategy 2 for CINA produce a linear amplification of ssDNA forward strand target.
  • an optional step of Strategy 2 described as Part C can be implemented to generate amplified dsDNA target sequence.
  • a Primer 2 (P2) is included in Part C.
  • Part C may be performed simultaneously or sequentially with Part B.
  • P2 is complementary to and bind to ssDNA1 from Part A.
  • ssDNA1 and ssDNA2 are complementary in a partial overlap and can form a staggered duplex.
  • P2 bind to ssDNA 1 and DNA polymerase extends the 3’ end of P2, regenerating Cas Nick-Site 2 and amplifying until reaching Nick-Site 1.
  • DNA polymerase displaces ssDNA2 and regenerates Cas Nick-Site 2 for the next round of nicking.
  • Cas nicks the regenerated Cas Nick-Site 2 and DNA polymerase re-enters at the newly synthesized nick site and displaces the bottom strand (ssDNA 4 ), regenerating the dsDNA template for the next cycle of nicking.
  • ssDNA4 is complementary to ssDNA3 formed as described in Part B.
  • Products from Part C include ds-gDNA containing regenerated PAM 2, Nick- Site 2, and target sequence.
  • FIG.23 shows a diagram of a portion of pUC19 plasmid sequence and labeled sites. A 502 bp section of pUC19 was cut up for use in demonstrating CINA Strategy 2. Amplification products shown in FIG.23 include ssDNA1 (280 b), ssDNA2 (402 b), dsDNA2 (280 bp), and ssDNA3 (180 b). Bases (b). Base pairs (bp). Primers shown in FIG.23: forward primer (p19 FP500) and reverse primer (p19 RP500).
  • FIG.24A shows reaction products run on 10% Native PAGE.
  • FIG.24B shows reaction products run on 1.5% agarose gel electrophoresis.
  • Lanes for both FIG.24A and FIG.24B are as follows: C. DNA only (500 bp dsDNA) 1.
  • 502 Nick 1 Cas9n + sgRNA targeting 502 Nick 1 site 2.
  • Nick B2 Cas9n + sgRNA targeting 502 Nick B2 site 5.
  • Nick B2 + Bst 3.0 + p19 FP500 [000203]
  • L2 ssDNA 1 (280 b) is displaced upon addition of Bst 3.0 which is observed in native PAGE but not agarose gels.
  • L3 RP-500 transforms ssDNA 1 to dsDNA 2 (280bp) as observed in both native PAGE and agarose gels.
  • L5 ssDNA 2 (402 b) is successfully displaced upon addition of Bst 3.0 which is observed in native PAGE but not agarose gels.
  • L6 FP-500 transforms ssDNA 2 to dsDNA 3 (280bp) as observed in both native PAGE and agarose gels.
  • Two independently labelled probes bind to unlabeled ssDNA 1.
  • a FAM probe and a Biotinylated probe bind to different regions of unlabeled ssDNA 1.
  • the probes can then bind to FITC antibody and streptavidin, respectively. This permits detection of our unlabeled amplicon by LFA.
  • CINA Strategy 2 theorizes that adding 2 primers that can bind upstream of 2 nick sites can create exponential amount of dsDNA strands.
  • CINA Strategy 2 Part A+B+C theorizes that with 2 nick sites, the polymerase displaces 2 ssDNAs: ssDNA 1 and ssDNA 2.
  • ssDNA 1 binds Primer 2 to create dsDNA 2 and recreates Nick Site 2 which can now get nicked again and polymerase can displace ssDNA 3.
  • ssDNA 2 binds Primer 1 to create dsDNA 3 and recreatesnick Site 1 which can now get nicked again and polymerase can displace ssDNA 4.
  • ssDNA 3 and 4 can create dsDNA 4.
  • L2 ssDNA 1 (280 b) is displaced upon addition of Bst 3.0 after 502-Nick1 which can be detected on LFA using Probes 1 and 2.
  • L3 RP500 binds to ssDNA1 to make dsDNA2 (280bp) as observed in agarose gels and therefore cannot be detected by LFA anymore.
  • L5 ssDNA 2 (402 b) is displaced upon addition of Bst 3.0 after NickB2 which cannot be detected on LFA using Probes 1 and 2, showing specificity of the probes.
  • L6 FP500 binds to ssDNA2 to make dsDNA3 (402bp) as observed in agarose gels.
  • L8 Polymerase binds to 502 Nick 1 and Nick B2 and respectively displaces ssDNA 1 (280 b) and ssDNA 2 (402 b). ssDNA1 can be detected by probes 1 and 2 with LFA.
  • L9 RP500 binds to displaced ssDNA1 (280b) to make dsDNA2 (280 bp) which regenerates NickB2 site, which can then produce ssDNA 3 (180b).
  • FIG.26A The gel image in FIG.26A was further analyzed to quantitate amplicon production by band quantification.
  • FIG.26C is gel image illustrating the amplicon bands measured for intensity. Boxed numbers inside gel image correspond to order in which lanes were selected for analysis .
  • Band 1 adjusted density (template band) is graphed in L5 and L6 in columns 1. Note the exponential amplification of dsDNA 3 in L6 compared to ssDNA 3 in L5.
  • dsDNA 3 adjusted density L6 1.5584.
  • ssDNA 1 and ssDNA 2 are complementary in the middle section and may form a staggered duplex through base pairing (FIG.42).
  • Primer 1 (P1) as shown in FIG.41 can bind near the 3’ end of ssDNA2 and use this as a template for extension with Klenow fragment (-exo).
  • Klenow extension displaces ssDNA 1 and also regenerates Cas9n D10A Nick Site 1 for a subsequent round of nicking.
  • Klenow can reenter and at the newly synthesized nick site and displace the top strand (ssDNA3), regenerating the dsDNA template for the next round of nicking.
  • FIG.43 version 1 and version 2 of Primer 1 are shown.
  • Primer 1 Version 2 (P1 V2) contains a promoter sequence which when incorporated into ssDNA3 will allow for production of mRNA transcripts from the promoter site.
  • FIG.44A shows a map with dsDNA produced after P1 V2 extension.
  • FIG.44B shows a sequence of ssDNA 3 produced following Nick 3 cleavage and P1 V2 extension. ssDNA 1 , ssDNA 2, and ssDNA3 can be detected and quantified by probes.
  • Part C of CINA Strategy 2 primary target in the form of dsDNA can be produced.
  • Primer 2 (as shown in FIG.45), In Part B of ssDNA 1 and ssDNA 2 are complementary in a middle section and may form a staggered duplex through base pairing.
  • Primer 2 (P2) binds to the 3’ end of ssDNA1. Klenow extends to the 3’ using ssDNA1 as template. This regenerates Nick Site 2.
  • Cas9n D10A targeting Nick Site 2 nicks the newly synthesizes strands allowing for a subsequent round of Klenow extension from regenerated Nick Site 2 while displacing the bottom strand.
  • FIG.46 shows a map of puc19 DNA sequence with the positions of excised dsDNA, ssDNA2 from Nick Site 2, ssDNA1 from Nick Site 1, and Primer 1 location.
  • dsDNA4 can be detected and quantified through standard techniques of double strand DNA detection and analysis (e.g., agarose gel electrophoresis and labeling with a dsDNA-specific dye such as SYBR TM Green).
  • Strategy 2-B for amplification of mouse Exo1.
  • this example includes a description of amplification of a portion of the Exonuclease 1 (Exo1) gene from Mus musculus using Strategy 2.
  • the following parameters are utilized.
  • a portion of genomic DNA from Mus musculus containing the Exo1 target sequence was chosen. This region of genomic DNA sequence is included within Mus musculus BAC clone RP24-531A1 (GenBank: AC153644.3).
  • Table 6 lists sequences for this CINA method. Table 6: Mouse Exo1 CINA primer sequence, reaction products, and template WSGR Docket No.
  • ssDNA 1 and ssDNA 2 form a staggered duplex: 3’-AAGAACATTCTTCGGGGACAGAGGTCAGTTCCTGTTATAGGTTGAGTGAGGTCTCTGTCTTCTACTCTAGA 5’-TGTGTACGCGCTCAGAGCAATATTTCACTAAGTGCAGACCGTTGCCTACAAGAAA AATTGTTCGGGCTCACACATGCGCGAGTCTCTCGTTATAAAGTGATTCACGTCTGGCAACGGATGTTCTTT ATCTTATCAACTGGAGAGATGTATTAGTCTCAATGTCATTTTAAAT-3’ TAGAATAGTTGACCTCTCTACATAATCA-5’ WSGR Docket No.
  • ssDNA1, ssDNA2, and ssDNA3 can be detected and quantified by probes.
  • ssDNA1, ssDNA2, and ssDNA3 can be detected and quantified by probes.
  • Example 3 TGTGTACGCGCTCAGAGAGCAATATTTCACTAAGTGCAGACCGTTGCCTACAAGAAAATCTTATCAACTGG AGAGATGTATTAGT-3’.
  • nicking primers a 790 bp template target sequence from pUC19 was used and a diagram of the region is shown in FIG.27.
  • Primers that bind on nick site are termed as nicking primers (nP). Primers not binding on nicking site are indicated. Forward Nicking Primers are indicated. Reverse Nicking Primers are indicated.
  • Nicking target sequence is indicated.
  • nP nicking primers
  • Lane 4 primers P1S2 generate dsDNA 1, substrate for Cas9n which generate more intense band of ssDNA.
  • Lane 3 primer Fp500 regenerates 502 Nick 1, as increased band intensities are observed for both 568b & 180b products. Lanes 5 & 7, primers P1S2 and P2S1 regenerate Nick1, as increased band intensities are observed for both 568b & 180b products. Lane 2, Lane 4 and Lane 7, ssDNA formation observed in LFA. [000236] Conclusion: Primers P1S2 and P2S1 can successfully bind ssDNA displaced by Bst 3.0 polymerase and convert them into dsDNA. Positive LFA results in lanes 2, 4 & 7 indicate P2S1 primers generate dsDNA and Cas9n subsequently displaces ssDNA.
  • Lane 2 502Nick1 &NickB2v2 and their nick regenerating primers produce 152bp product.
  • Conclusion Three different nicking primers generate band of interest. Three different set of nick regenerating primer can amplify and generate corresponding CINA products.
  • FAM labelled nicking primers bind on nicking sites to regenerate nick sites and can be detected on gels using FAM fluorescence channel. FAM exposure of an agarose gel following electrophoresis was used in FIG.33B to detect FAM labelled dsDNA CINA amplicons. [000245] Observations: Lane 4 and lane 8 (Left gel), FAM labelled forward, and reverse primers generated dsDNA visualized by FAM exposure. Post staining by Gel Red was used to confirm the position of dsDNAs. [000246] Conclusion: BHQ1-FAM dual labeled can be used for confirmation of products formation.
  • FAM labelled product formation establish the generation of ssDNA from nick site and conversion to dsDNA by primer and polymerase.
  • Primer design according to FIG.27 was used here.
  • Primer length tested 27, 33 and WSGR Docket No. 58557-707.601 36b.
  • Reaction products are analyzed by agarose gel electrophoresis and DNA labeling. Results are shown in FIG.34.
  • [000248] Observation: Lane 3, 4 and 5, all 27b, 33b and 36b FP converted the ssDNA1 to dsDNA2.
  • CINA reaction optimzation [000250] CINA reaction parameters were varied to determine the effect on amplicons efficiency of CINA amplicon products. [000251] Effect of gp32 (a single-strand binding protein) in CINA amplification. Addition of 4 ⁇ M GP32 was found to increase the intensity of linear (LIN) DNA band formation. Addition of gp32, increases the overall amplification and produced more intense bands by Bst polymerase. [000252] Temperature: [000253] Reaction temperature was varied in CINA reactions using Cas9n to determine effect on amplification. The range of reaction temperatures tested was from 25°C - 65°C. Individual test temperatures: 25°C, 37°C, 45°C, 55°C, and 65°C.
  • Cas9n activity was tested. Both Cas9n-502Nick1 and NickB2v2 complex were found to generate nicks over a wide range of temperatures from 25°C to 65°C. At 37 °C, the vast majority of supercoiled plasmid DNA was converted to Nicked DNA. Conclusion: Cas9n active on wide range of temperature from 25°C and 65°C. Next, the effect of temperature on CINA amplicon production was tested. Primer design according to FIG.27 was used here. As shown in agarose gel following electrophoresis in FIG.35, CINA amplification of expected amplicons was observed over the temperature range of 25°C, 37°C and 45°C. CINA may be effective in room temperature under optimized conditions.
  • Bst 3.0 Isothermal amplification buffer (IAB2): 150 mM KCl, 20 mM Tris-HCl, 0.1% Tween ⁇ 20, 2 mM MgSO4, 10 mM (NH4)2SO4, pH 8.8 @ 25°C.
  • IAB2 Isothermal amplification buffer
  • +502 FP/RP Addition of 502 primers generate two specific products of expected sizes in IAB2 (L2), but with decreased intensity in r3.1 (L1). [000260] Conclusions: Amplification is more efficient in buffer IAB2 than in buffer r3.1. 502 FP & RP seem to generate specific amplicons in buffer IAB2 (L2). [000261] Optimization of Ratio and Concentrations of Cas enzyme:sgRNA:DNA template [000262] Next, experiments were conducted to test and optimize Cas9n:sgRNA:DNA molar ratios & concentrations. Primer design according to FIG.27 was used here.
  • a second strategy tested was LFA with FAM-labelled and biotin-labelled primers.
  • two independently labelled probes are illustrated binding to unlabeled ssDNA 1.
  • a FAM probe and a Biotinylated probe bind to different regions of unlabeled ssDNA 1.
  • the probes can then bind to FITC antibody and streptavidin, respectively. This permits detection of our unlabeled amplicon by LFA.
  • the following experiment was conducted. Amplicons were generated using both nicking & non-nicking primers for strategy 1 according to the CINA design in FIG.27.
  • BHQ1-FAM dual labeled can be used for confirmation of products formation and thereby can be used in real-time CINA detection.
  • Expected products include 1) 568 bp amplicon; 2) 386 bp amplicon, and the final reaction product 3) 180 bp amplicon.
  • FAM exposure of products run on an agarose gel in FIG.33C show direct detection of final amplicon products.
  • Gel Red post- staining of the gel confirmed the location of the DNA bands corresponding to the expected amplicons.
  • Image J was used to measure band intensity of FAM labeled amplicons from the gel in FIG.33C and results were graphed in FIG.33D measuring an increase in relative intensity of CINA bands compared to the corresponding location in the NTC lane.
  • final CINA amplicon product (180 bp) shows as larger increase in relative intensity indicating that as the end product of a CINA reaction reaches a greater extent of amplification compared to non-end WSGR Docket No. 58557-707.601 product CINA amplicons and that the increase in amplification efficiency between CINA amplicons can be quantitated in directly-labeled amplicon products without a need for further labeling.
  • Example 6 Reaction conditions and reagents for use in Strategy 1 and Strategy 2 of CINA [000277] Both Strategy 1 and Strategy 2 of CINA are designed to operate as isothermal reactions without the need for thermocycling. Reaction conditions can be maintained at about a constant temperature equivalent to an ambient temperature.
  • Reaction conditions can also various according to an ambient temperature during the operation of the methods. Length of reaction time can vary depending on factors including efficiency of amplification, sensitivity of detection assay used to detect and quantify amplified nucleotide product, ambient temperature during reaction, starting quantity of material to be amplified, and the selection of Steps of Strategy 1 or Strategy 2 of CINA operated according to the desired amplified product.
  • An exemplary reaction mix for use in either Strategy 1 or Strategy 2 of CINA comprises 50 mM nCas9, 20 mM of Cas12a2, 500 nM of sgRNA1, 150 nM of sgRNA2, 300 nM of primer 1, 100 nM of primer 2, 50 ng/ ⁇ L of template nucleic acid containing target DNA, 25 mM Tris at pH 8.0, 20 mM NaCl, 40 mM MgCl2, 5 mM DTT. Reaction is carried out in presence of template at ambient temperature of about 25°C for about 30 minutes.
  • Exemplary reagents for use in the reaction mix for either Strategy 1 or Strategy 2 of CINA comprise 20-500 nM Cas nickase, 20-500 nM Cas DSB enzyme, 50-1000 nM of each sgRNAs, 50-500 ⁇ M dNTPs/NTPs, 10-1000 nM of each or either primers, 0.1-10 U/ ⁇ L of DNA polymerase, 10-500 mM of buffering agent such as Tris, pH 7-9, 10-500 mM MgCl 2 or MgSO 4 or MgAcetate.
  • Additional reagents can be 2-40 mM of reducing agents such as DTT, 20-500 mM of NaCl or KCl, 10-300 ⁇ g/mL of stabilizing agents such as BSA, 0.01-10% of crowding agent such as PEG, 0.1-2.0 X of fluorescent DNA-binding dye such as SYBR green, 10-1000 nM of gene-specific probe, 10-300 ⁇ g/mL of solubilizing agent, 0.001-1% of non-ionic detergent such as Triton-X100, 0.1-1U of nuclease inhibitor.
  • reducing agents such as DTT
  • stabilizing agents such as BSA
  • crowding agent such as PEG
  • 0.1-2.0 X of fluorescent DNA-binding dye such as SYBR green
  • 10-1000 nM of gene-specific probe 10-300 ⁇ g/mL of solubilizing agent
  • 0.001-1% of non-ionic detergent such as Triton-X100
  • Reagents 1.100 nM Cas9n 2.200 nM sgRNA 3.0.1 U RNase inhibitor 4. NEBuffer 3.1 5. pUC19 6. Proteinase K 7. Total Reaction volume 20 ⁇ l [000282] RNase free water, NEBuffer 3.1, 200nM sgRNA, and 100nM Cas9n are combined in a total reaction volume of 20 ⁇ L. After reaction is set up, reaction is incubated for 20 min at 37°C. [000283] Step 2: Digestion reaction. [000284] Add 10nM target DNA (pUC19 plasmid). Add 0.1U RNase inhibitor. Incubate at 37°C for 60 min.
  • Step 3 Inactivation of the reaction.
  • Step 4 Analysis by gel electrophoresis.
  • [000288] Run reaction products on a 1.5% agarose gel for sufficient time to separate products of different nucleotide lengths. Visualize and quantify products using a fluorescent dye (e.g., ethidium bromide, SYBR TM Green, SYBR TM Green II, or SYBR TM Gold).
  • Amplification reaction set up [000290] The reaction mixtures for Cas9nAR are prepared separately with 10 ⁇ L Part A solution and 10 ⁇ L Part B solution.
  • Part A solution is set up in a total reaction volume of 10 ⁇ L. This step includes the preincubation of sgRNA with Cas9n.
  • Reagents 1. RNase free water 2. NEBuffer 3.1 3.200nM sgRNA1 4.200nM sgRNA2 5.100nM Cas9n
  • Step 1 using Part A solution Reagents are assembled according to the above concentrations in 10 ⁇ L. Incubate for 20 min at 37°C.
  • Step 2 is set up in the Part A solution reaction following the completion of Step 1.
  • Step 2 Included in Step 2 are: 1x NEBuffer2, add 0.2U/ ⁇ L exo-klenow polymerase, add 0.1 U RNase inhibitor, and add 0.4X SYBR TM Green, 0.4X SYBR TM Green II, or 0.4X SYBR TM Gold.
  • Bst 2.0, Bst 3.0, or a combination of Bst 2.0 and Bst 3.0 can be used with an appropriate buffering system of the DNA polymerase.
  • WSGR Docket No. 58557-707.601 Part B solution is set up in a separate reaction from Part A in a total reaction volume of 10 ⁇ L.
  • 3 uL DNA template (pUC19 plasmid, 30nM) was added to each reaction.
  • 1 uL gp32 120uM
  • 0.75uL dNTPs 40mM
  • 1uL Bst 3.0 8U/uL
  • 1uL Bst 2.0 8U/uL
  • 1.2uL of primer 502nPrimer1 S1 (10uM) & 502nPrimer2 S1 (10uM) were included.
  • Samples were incubated at 25°C for 1 hour to allow for amplification. Following amplification, reactions were treated with 1uL Proteinase K (0.8U/uL) for 10 minutes at 25°C for termination.

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Abstract

La présente invention concerne des compositions pour l'amplification isotherme d'acides nucléiques ciblés par l'utilisation de réactifs CRISPR. La présente invention concerne également des procédés d'amplification isotherme de séquences d'acides nucléiques ciblées par l'utilisation de réactifs CRISPR.
PCT/US2024/019074 2023-03-08 2024-03-08 Compositions et procédés d'amplification isotherme d'acides nucléiques Pending WO2024187091A1 (fr)

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Citations (6)

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US20200199664A1 (en) * 2014-11-11 2020-06-25 Illumina, Inc. Polynucleotide amplification using crispr-cas systems
US20210207130A1 (en) * 2015-12-07 2021-07-08 Arc Bio, Llc Methods and compositions for the making and using of guide nucleic acids
US20210207203A1 (en) * 2018-06-26 2021-07-08 The Broad Institute, Inc. Crispr double nickase based amplification compositions, systems, and methods
US20210269866A1 (en) * 2018-06-26 2021-09-02 The Broad Institute, Inc. Crispr effector system based amplification methods, systems, and diagnostics
WO2022232425A2 (fr) * 2021-04-29 2022-11-03 Illumina, Inc. Techniques d'amplification pour la caractérisation d'acides nucléiques
US20230040148A1 (en) * 2019-02-22 2023-02-09 Integrated Dna Technologies, Inc. Lachnospiraceae bacterium nd2006 cas12a mutant genes and polypeptides encoded by same

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200199664A1 (en) * 2014-11-11 2020-06-25 Illumina, Inc. Polynucleotide amplification using crispr-cas systems
US20210207130A1 (en) * 2015-12-07 2021-07-08 Arc Bio, Llc Methods and compositions for the making and using of guide nucleic acids
US20210207203A1 (en) * 2018-06-26 2021-07-08 The Broad Institute, Inc. Crispr double nickase based amplification compositions, systems, and methods
US20210269866A1 (en) * 2018-06-26 2021-09-02 The Broad Institute, Inc. Crispr effector system based amplification methods, systems, and diagnostics
US20230040148A1 (en) * 2019-02-22 2023-02-09 Integrated Dna Technologies, Inc. Lachnospiraceae bacterium nd2006 cas12a mutant genes and polypeptides encoded by same
WO2022232425A2 (fr) * 2021-04-29 2022-11-03 Illumina, Inc. Techniques d'amplification pour la caractérisation d'acides nucléiques

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